Protein tyrosine phosphatase-prl-1 a a marker and therapeutic target for pancreatic cancer

ABSTRACT

Using gene expression profiling, the present Invention identifies Protein tyrosine phosphatase IVA member 1 (PRL-1) as a diagnostic marker and therapeutic target for pancreatic cancer. The Invention therefore provides methods for prediction and detection of PRL-1 associated cancers, and evaluation of inhibitors of PRL-1. The Invention also provides a method of treating or preventing pancreatic cancer in a subject.

The present application claims the benefit of co-pending U.S.Provisional Patent Application Ser. No. 60/486,231, filed Jul. 10, 2003,U.S. Provisional Patent Application Ser. No. 60/453,380, filed Mar. 10,2003, and U.S. Provisional Patent Application Ser. No. 60/451,488, filedMar. 3, 2003, the entire disclosures of which are specificallyincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of molecularbiology and cancer therapy. More particularly, it concerns diagnosticmarkers and drug targets for pancreatic cancer.

2. Description of Related Art

Pancreatic cancer is the fourth leading cause of cancer death amongadults in the United States. In the year 2000 alone, an estimated 28,300new cases of pancreatic cancer were diagnosed in the United States andnearly 28,200 patients were estimated to have died. Close to 90% ofpatients diagnosed with pancreatic cancer die within the first yearfollowing diagnosis. The deadliness of this disease has encouraged asearch for factors that influence incidence and the molecular eventsthat are involved in pancreatic tumor progression. At the molecularlevel, it is thought that the accumulation of defects in specific genesthat contribute to the growth and development of normal tissue areresponsible for the progression of cancer. Therefore, understanding theeffects of genetic lesions that are common in the development ofpancreas cancer will no doubt lead to new and more effective ways todiagnose, treat, and prevent this devastating disease.

The advent of cDNA microarray technology has made possible theidentification and validation of new potential targets for drugdevelopment and analysis of the secondary effects of agents bymonitoring changes in the expression of downstream genes. cDNAexpression microarray analysis allows for the rapid identification ofpotential targets for drug development by examining the expression ofthousands of genes in cancer cells versus normal cells. The changes ingene expression patterns from normal to tumor cells provide a backgroundto determine what pathways are altered in cancer cells on acomprehensive scale.

Although some genes have been identified that are involved in pancreaticcancer, these discoveries have not proved beneficial in advancing thetreatment and prevention of this disease. Thus, there still exists aneed for additional disease markers and therapeutic targets in the fieldof pancreatic cancer.

SUMMARY OF THE INVENTION

The present invention addresses the deficiencies in the art of anefficacious therapy for treating pancreatic cancer by investigating themolecular basis of the disease. In comparing pancreatic cancer cells tothat of normal pancreas, by expression profiling, protein tyrosinephosphatase IVA member 1 (PRL-1) was identified as a diagnostic markerand a therapeutic target in treating this disease. Thus, the presentinvention provides a method of diagnosing or predicting development ofpancreatic cancer in a subject comprising (a) obtaining a pancreaticcell sample from the subject; and (b) assessing PRL-1 activity orexpression in the cell, wherein increased activity or expression ofPRL-1 in the cell, when compared to a normal cell of the same type,indicates that the subject has or is at risk of developing pancreaticcancer.

A pancreatic cell sample embodied in the present invention may beprecancerous pancreatic cell sample, a metastatic pancreatic cellsample, or a malignant pancreatic cell sample. Malignant pancreatic cellsamples may further comprise a ductal adenocarcinoma cell sample, anintraductal papillary neoplasm cell sample, a papillary cystic neoplasmcell sample, a mucinous cystadenocarcinoma cell sample, a mucinouscystadenoma cell sample, an acinar carcinoma cell sample, anunclassified large cell carcinoma sample, a small cell carcinoma sample,or a pancreatoblastoma cell sample.

In other embodiments, the cell is a pancreatic tumor cell.

In a particular embodiment, the present invention comprises assessingPRL-1 expression or activity in a cell or sample, such as a tissuesample, by Northern blotting, quantitative RT-PCR, Western blotting orquantitative immunohistochemistry.

In some embodiments, the subject has previously been diagnosed withcancer or the subject has not previously been diagnosed with cancer andappears cancer free at the time of testing. In another embodiment, thepresent invention comprises administering a prophylactic cancertreatment, or a cancer therapy to the subject following testing. Inother embodiments, the cancer therapy may be a chemotherapy, aradiotherapy, an immunotherapy, a gene therapy, a hormonal therapy orsurgery.

In still another embodiment, the present invention provides a method ofpredicting the efficacy of a pancreatic cancer therapy comprising (a)administering a cancer therapy to the subject; (b) obtaining apancreatic tumor cell sample from the subject; and (c) assessing PRL-1activity or expression in the tumor cell of the sample, whereindecreased activity or expression of PRL-1 in the tumor cell, whencompared to a tumor cell of the same type prior to treatment, indicatesthat the therapy is efficacious.

In further embodiments the present invention comprises assessing PRL-1expression comprising measuring PRL-1 protein levels, or measuring PRL-1transcript levels. In other embodiments, the present invention furthercomprises assessing PRL-1 activity or expression at multiple timepoints.

In still yet another embodiment, the present invention comprises amethod of screening a candidate compound for anti-cancer activitycomprising (a) providing a pancreatic cancer cell; (b) contacting thecell with a candidate compound; and (c) assessing the effect of thecandidate compound on PRL-1 expression or activity, wherein a decreasein the amount of PRL-1 expression or activity, as compared to the amountof PRL-1 expression or activity in a similar cell not treated with thecandidate compound, indicates that the candidate compound hasanti-cancer activity.

The candidate compound of the present invention may be a protein, anucleic acid or an organo-pharmaceutical.

In some embodiments of the invention the tumor cell may be selected fromthe group consisting of a precancerous pancreatic cell, a metastaticpancreatic cell, or a malignant pancreatic cell. The malignantpancreatic cell may further comprise a ductal adenocarcinoma cell, anintraductal papillary neoplasm cell, a papillary cystic neoplasm cell, amucinous cystadenocarcinoma cell, a mucinous cystadenoma cell, an acinarcarcinoma cell, an unclassified large cell carcinoma, a small cellcarcinoma, or a pancreatoblastoma cell.

In further embodiments, a method of treating cancer comprisesadministering to a subject in need thereof a composition that inhibitsPRL-1 activity or expression.

In still further embodiments, the candidate compound may be a protein, anucleic acid or an organo-pharmaceutical. In yet a further embodiment,the protein is an antibody that binds immunologically to PRL-1. In stillyet a further embodiment, the nucleic acid may be a PRL-1 antisensenucleic acid, a PRL-1 RNAi nucleic acid, or an antibody encoding asingle-chain antibody that binds immunologically to PRL-1.

In some embodiments, the invention further comprises administering asecond cancer therapy such as a chemotherapy, a radiotherapy, animmunotherapy, a gene therapy, a hormonal therapy or surgery to thesubject.

In further embodiments, the composition of the invention may beadministered more than once.

In a further embodiment, the present invention provides a method ofdiagnosing or predicting development of pancreatic cancer in a subjectcomprising subjecting the subject to whole body scanning for PRL-1activity or expression in a cell.

In still a further embodiment, the present invention provides a methodof monitoring an anticancer therapy comprising assessing the expressionor function of PRL-1 in a pancreatic cancer cell of a subject followingor during provision of the anticancer therapy.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Schematic of gene expression profiling using a microarray.

FIG. 2. A hybridization of gene expression from BXPC-3 pancreatic cancercells versus a Hela cell reference using the 5,760 gene chip.

FIGS. 3A-3C. Overexpression of genes identified from the microarrayanalysis. FIG. 3A—RT-PCR of various genes identified from the microarrayanalysis. FIG. 3B—RT-PCR of pancreatic cell lines overexpressing PRL-1.FIG. 3C—RT-PCR of pancreatic tumor samples showing overexpression ofPRL-1.

FIG. 4. Pancreatic cancer tissue array. To aid in the validation ofpotential new targets for drug development, a pancreatic cancer tissuearray was constructed consisting of 50 pancreatic cancer spots and 20normal pancreas spots.

FIGS. 5A-5D. FIG. 5A—Antisense inhibitor AS-Prl-1C reduced mRNA levelsof PRL-1. FIG. 5B—Real Time PCR data verifies that antisense oligo Ctargets PRL-1. FIG. 5C—Treatment of pancreatic cancer cells (MiaPaCa-2)with AS-Prl-1C results in arrest of cell growth. FIG. 5D—Pancreaticcancer cells (MiaPaCa-2) treated with AS-Prl-1C show a dramatic increasein apoptosis.

FIG. 6. Identification of siRNA sequences that reduced PRL-1 expression.

FIGS. 7A-7C. FIG. 7A—Western blot detection of His-tagged PRL-1 proteinin TNT mixture. FIG. 7B—TNT product increased phosphatase activity. FIG.7C—Inhibitory activity of tyrosine phosphatase inhibitors.

FIG. 8. Anti-PRL-1 activity of inhibitors.

FIG. 9. Clustal W alignment shows sequence identity and similaritybetween PRL-1 and the human phosphatases SHP2 and PTEN. The sequencealignment shows high homology in the active site of the phosphatasedomains and increased variation outside of the active sites.

FIG. 10. 3D model of PRL-1 based on PTEN. The homology model of PRL-1was constructed based on the above structure alignment using themodeling software in INSIGHT II. The PRL-1 homology model indicated ahighly conserved hydrophobic core, but a changed specificity pocketwithout any major distortion of the geometry of the active site.

FIG. 11. Docking models of PRL-1 compounds.

FIG. 12. Lipid phosphatase activity of PRL-1.

FIGS. 13A-13C. Inhibition of cell proliferation by PRL-1 inhibitorsusing a SRB staining assay. FIG.13A—Inhibition of MiaPaCa-2 cell growthby UA668394. Cells were exposed to different doses of UA668394 (0.2 μMto 200 μM) for four days by SRB (Sulforhodamine B) staining. Theestimated IC₅₀ is 1.2 μM. FIG. 13B—Inhibition of cell proliferation inpancreatic cancer cells Panc-1 and Mia PaCa-2 by the compound UA66839-1analog. FIG. 13C—Inhibition of cell proliferation in pancreatic cancercells Panc-l and Mia PaCa-2 by the compound UA668394-2 analog.

FIG. 14. Inhibition of PRL-1 expression by SMARTPool siRNA. MiaPaCa-2cells were transiently transfected with either 50 nM (Lanes 1 and 6),100 nM (Lanes 2 and 7) or 200 nM (Lanes 3 and 8) of the PRL-1 siRNAoligo mixture and harvested at either 48 hours (Lanes 1, 2 and 3) or 72hours (Lanes 6, 7 and 8) after transfection. Lanes 4 and 9 control for48 hour and 72 hour treatments, respectively. Lanes 5 and 10 are notreatment control (no siRNA and no Lipofectin) for 48 hour and 72 hourtime points, respectively.

FIG. 15. PTEN Assay

FIG. 16. Mia PaCa-2 cells treated with the UA668394 compound was foundto have an IC₅₀ of 1.2 μM. Cells treated with the UA19999 and UA45336compounds showed an IC₅₀ of 120 μM and 95 μM respectively.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

A. The Present Invention

As discussed above, one of the most deadly cancers is pancreaticcancers, with few patients living more than one year past initialdiagnosis. Despite considerable focus on this disease, the prognosis forpatients remains poor. Thus, intense research must be focused on cancersof the pancreas.

One aspect of this research is the search for a molecular basis forpancreatic cancer. The present inventors sought to examine theexpression profiles of pancreatic cancer cells and compared these tonormal cells. In so doing, they identified a group of dysregulatedgenes, the expression of which is greater or less in cancer cells thanin a corresponding non-cancerous cell.

One of these genes, PRL-1, was highly overexpressed in most pancreaticcancer cells examined. Overexpression of the PRL-1 gene in pancreaticcancer cell lines was confirmed using Northern blotting and RT-PCR. Tofurther ascertain that PRL-1 is a viable molecular target for cancertherapy, antisense oligonucleotides were used to inhibit the expressionof PRL-1 in pancreatic cancer cells. Treated cells showed a significantincrease in apoptosis and a decrease in accumulation of cells in the Sphase. From these results, PRL-1 was confirmed as a diagnostic markerfor pancreatic cancer, and a therapeutic target in treating thisdisease.

Thus, the present invention provides methods of assessing the activityor expression of PRL-1 protein or transcripts levels using a variety oftechniques, the goal being the identification of cancers of pancreaticorigin. The present invention also provides methods of screening forcandidate inhibitors of PRL-1. Finally, the present invention providesmethods of treating a cancer, in particular pancreatic cancer, byproviding compositions that inhibit PRL-1 activity or expression, eitheras a single agent or in combination with other therapeutic agents. Thedetails of the invention will be provided in the following materials.

B. Protein Tyrosine Phosphorylation and PRL-1 Phosphorylation ofcellular proteins, particularly tyrosine phosphorylation, plays acentral role in the regulation of a number of cellular processes,including cellular proliferation and differentiation (Tonks, 1993;Pawson et al., 1994). The protein tyrosine phosphatases (PTPase) belongto the protein phosphatase gene family. This phosphatase family consistsof phosphatases that remove phosphate groups from protein tyrosineresidues with high selectivity. One phosphorylated tyrosine residue mayserve as a substrate, but another phosphotyrosine residue of the sameprotein may not. These phosphatases exist in a wide range of sizes andstructural forms including transmembrane receptor-like andnon-transmembrane forms. However, they all share homology within aregion of 240 residues which defines a catalytic domain and contains a(I/V)HCXAGXXR(S/T)G consensus amino acid sequence near the C-terminus.Mutation of the active site cysteine residue abolishes this activity.

One member of this family of protein phosphatases is protein tyrosinephosphatase IVA member 1 (PRL-1), a non-transmembrane proteinphosphatase. PRL-1 is a unique nuclear tyrosine phosphatase thatcontrols cell growth. PRL-1 is 20 kDa in size, and is distinct fromother protein tyrosine phosphatases of this family. PRL-1 has littlehomology to other PTPases outside the active site. However, PRL-1 isclosely related to two other protein tyrosine phosphatases, PRL-2 andPRL-3. These PRL phosphatases contain a consensus motif for proteinprenylation at the C-terminus (Zeng et al., 1998).

PRL-1 was initially identified as an immediate early gene involved inregenerating the liver (Diamond et al., 1996). This gene was also foundto be expressed in mitogen-stimulated fibroblast. Stably transfectedcells which overexpress PRL-1 demonstrate altered cellular growth andmoiphology and a transformed phenotype. The expression of PRL-1 isassociated with cell proliferation and differentation due to its abilityto regulate the protein tyrosine phosphorylation and dephosphorylationof substrates that remain unknown. Overexpression of PRL-1 in epithelialcells has been shown to result in tumor formation in nude mice (Cates etal., 1996). It has also been suggested that PRL-1 function is regulatedin a cell cycle dependent manner. PRL-1 has also been implicated inregulating progression through mitosis, possibly by modulating spindledynamics (Wang et al., 2002). PRL-1 has been shown to be expressed in anumber of tumor cell lines (Wang et al., 2002). Thus, the art suggeststhat PRL-1 has diverse roles in various tissues. At a minimum, itappears that PRL-1 is important in normal cellular growth control andmay contribute to the tumorigenicity of some cancer cells (Diamond etal, 1994). The emergence of phosphatases, specifically protein tyrosinephosphatases, as potential therapeutic targets arose from recent studieswith targeting PTP1B. Knockout, antisense and drug development studieshave shown that down-regulation of PTP1B may be a good approach fortreating diabetes and obesity (Elcheby et al., 1999). In cancer, severalPTPs (e.g., PTP-a, PTP-E, Sapl, GLEPPI, PTP1 B) have been postulated todephosphorylate and activate proto-oncogene Src-family kinases. PRL-3and Cdc25B are other PTPs that have been shown to be specificallyup-regulated in various tumor types.

C. Prognostic and Diagnostic Methods

A variety of methods known to those of ordinary skill in the art areavailable for assessing the activity or expression of a gene product ina cell, tissue sample or organism. The present invention embodiesdiagnostic methods and methods for assessing PRL-1 activity orexpression comprising measuring PRL-1 protein or transcript levels.Methods of assessing for PRL-1 enzyme activity, or protein expressionlevels may also be employed. These methods are provided to identifysubjects who both may be at risk for developing cancer, and who alreadyhave pancreatic cancer. In addition, these same methods may be appliedto assess the efficacy of a cancer therapy.

Assays to assess the level of expression of a polypeptide are also wellknown to those of skill in the art. This can be accomplished also byassaying for PRL-1 mRNA levels, mRNA stability or turnover, as well asprotein expression levels. It is further contemplated that anypost-translational processing of PRL-1 may also be assessed, as well aswhether it is being localized or regulated properly. In some cases anantibody that specifically binds PRL-1 may be used. Assays for PRL-1activity also may be used.

1. Northern Blotting Techniques The present invention employs Northernblotting in assessing the expression of PRL-1 in a cancer or tumor cell.The techniques involved in Northern blotting are commonly used inmolecular biology and are well known to one of skilled in the art. Thesetechniques can be found in many standard books on molecular protocols(e.g., Sambrook et al., 2001). This technique allows for the detectionof RNA i.e., hybridization with a labeled probe.

Briefly, RNA is separated by gel electrophoresis. The gel is thencontacted with a membrane, such as nitrocellulose, permitting transferof the nucleic acid and non-covalent binding. Subsequently, the membraneis incubated with, e.g., a chromophore-conjugated probe that is capableof hybridizing with a target amplification product. Detection is byexposure of the membrane to x-ray film or ion-emitting detectiondevices.

U.S. Pat. No. 5,279,721, incorporated by reference herein, discloses anapparatus and method for the automated electrophoresis and transfer ofnucleic acids. The apparatus permits electrophoresis and blottingwithout external manipulation of the gel and is ideally suited tocarrying out methods according to the present invention.

2. Quantitative RT-PCR

The present invention also employs quantitative RT-PCR in assessing theexpression or activity of PRL-1 in a cancer or tumor cell. Reversetranscription (RT) of RNA to cDNA followed by relative quantitative PCR™(RT-PCR) can be used to determine the relative concentrations ofspecific mRNA species, such as a PRL-1 transcript, isolated from a cell.By determining that the concentration of a specific mRNA species varies,it is shown that the gene encoding the specific mRNA species isdifferentially expressed

In PCR™, the number of molecules of the amplified target DNA increase bya factor approaching two with every cycle of the reaction until somereagent becomes limiting. Thereafter, the rate of amplification becomesincreasingly diminished until there is not an increase in the amplifiedtarget between cycles. If one plots a graph on which the cycle number ison the X axis and the log of the concentration of the amplified targetDNA is on the Y axis, one observes that a curved line of characteristicshape is formed by connecting the plotted points. Beginning with thefirst cycle, the slope of the line is positive and constant. This issaid to be the linear portion of the curve. After some reagent becomeslimiting, the slope of the line begins to decrease and eventuallybecomes zero. At this point the concentration of the amplified targetDNA becomes asymptotic to some fixed value. This is said to be theplateau portion of the curve

The concentration of the target DNA in the linear portion of the PCR™ isdirectly proportional to the starting concentration of the target beforethe PCR™ was begun. By determining the concentration of the PCR™products of the target DNA in PCR™ reactions that have completed thesame number of cycles and are in their linear ranges, it is possible todetermine the relative concentrations of the specific target sequence inthe original DNA mixture. If the DNA mixtures are cDNAs synthesized fromRNAs isolated from different cells, the relative abundances of thespecific mRNA from which the target sequence was derived can bedetermined for the respective tissues or cells. This directproportionality between the concentration of the PCR™ products and therelative mRNA abundances is only true in the linear range portion of thePCR™ reaction.

The final concentration of the target DNA in the plateau portion of thecurve is determined by the availability of reagents in the reaction mixand is independent of the original concentration of target DNA.Therefore, the first condition that must be met before the relativeabundances of a mRNA species can be determined by RT-PCR for acollection of RNA populations is that the concentrations of theamplified PCR™ products must be sampled when the PCR™ reactions are inthe linear portion of their curves.

The second condition that must be met for an RT-PCR study tosuccessfully determine the relative abundances of a particular mRNAspecies is that relative concentrations of the amplifiable cDNAs must benormalized to some independent standard. The goal of an RT-PCR study isto determine the abundance of a particular mRNA species relative to theaverage abundance of all mRNA species in the sample. In such studies,mRNAs for β-actin, asparagine synthetase and lipocortin II may be usedas external and internal standards to which the relative abundance ofother mRNAs are compared.

Most protocols for competitive PCR™ utilize internal PCR™ internalstandards that are approximately as abundant as the target. Thesestrategies are effective if the products of the PCR™ amplifications aresampled during their linear phases. If the products are sampled when thereactions are approaching the plateau phase, then the less abundantproduct becomes relatively over represented. Comparisons of relativeabundances made for many different RNA samples, such as is the case whenexamining RNA samples for differential expression, become distorted insuch a way as to make differences in relative abundances of RNAs appearless than they actually are. This is not a significant problem if theinternal standard is much more abundant than the target. If the internalstandard is more abundant than the target, then direct linearcomparisons can be made between RNA samples.

The discussion above describes the theoretical considerations for anRT-PCR assay for clinically derived materials. The problems inherent inclinical samples are that they are of variable quantity (makingnormalization problematic), and that they are of variable quality(necessitating the co-amplification of a reliable internal control,preferably of larger size than the target).

Both of the foregoing problems are overcome if the RT-PCR is performedas a relative quantitative RT-PCR with an internal standard in which theinternal standard is an amplifiable cDNA fragment that is larger thanthe target cDNA fragment and in which the abundance of the mRNA encodingthe internal standard is roughly 5-100 fold higher than the mRNAencoding the target. This assay measures relative abundance, notabsolute abundance of the respective mRNA species.

Other studies are available that use a more conventional relativequantitative RT-PCR with an external standard protocol. These assayssample the PCR™ products in the linear portion of their amplificationcurves. The number of PCR™ cycles that are optimal for sampling must beempirically determined for each target cDNA fragment. In addition, thereverse transcriptase products of each RNA population isolated from thevarious tissue samples must be carefully normalized for equalconcentrations of amplifiable cDNAs. This is very important since thisassay measures absolute mRNA abundance. Absolute mRNA abundance can beused as a measure of differential gene expression only in normalizedsamples. While empirical determination of the linear range of theamplification curve and normalization of cDNA preparations are tediousand time consuming processes, the resulting RT-PCR assays can besuperior to those derived from the relative quantitative RT-PCR with aninternal standard. One reason for this is that without the internalstandard/competitor, all of the reagents can be converted into a singlePCR™ product in the linear range of the amplification curve, increasingthe sensitivity of the assay. Another reason is that with only one PCR™product, display of the product on an electrophoretic gel or some otherdisplay method becomes less complex, has less background and is easierto interpret.

3. Immunohistochemistry

The present invention also employs quantitative immunohistochemistry inassessing the expression of PRL-1 in a cancer or tumor cell.

Briefly, frozen-sections may be prepared by rehydrating 50 ng of frozen“pulverized” tumor at room temperature in phosphate buffered saline(PBS) in small plastic capsules; pelleting the particles bycentrifugation; resuspending them in a viscous embedding medium (OCT);inverting the capsule and pelleting again by centrifugation;snap-freezing in −70° C. isopentane; cutting the plastic capsule andremoving the frozen cylinder of tissue; securing the tissue cylinder ona cryostat microtome chuck; and cutting 25-50 serial sections containingan average of about 500 remarkably intact tumor cells.

Permanent-sections may be prepared by a similar method involvingrehydration of the 50 mg sample in a plastic microfuge tube; pelleting;resuspending in 10% formalin for 4 h fixation; washing/pelleting;resuspending in warm 2.5% agar; pelleting; cooling in ice water toharden the agar; removing the tissue/agar block from the tube;infiltrating and embedding the block in paraffin; and cutting up to 50serial permanent sections.

4. Western Blotting

The present invention also employs the use of Western blotting(immunoblotting) analysis to assess PRL-1 activity or expression in acell such as a pancreatic cancer cell. This technique is well known tothose of skill in the art, see U.S. Pat. No. 4,452,901 incorporatedherein by reference and Sambrook et al. (2001). In brief, this techniquegenerally comprises separating proteins in a sample such as a cell ortissue sample by SDS-PAGE gel electrophoresis. In SDS-PAGE proteins areseparated on the basis of molecular weight, then are transferring to asuitable solid support, (such as a nitrocellulose filter, a nylonfilter, or derivatized nylon filter), followed by incubation of theproteins on the solid support with antibodies that specifically bind tothe proteins. For example, in the present invention, anti-PRL-1antibodies specifically bind to PRL-1 proteins on the solid support.These antibodies may be directly labeled or alternatively may besubsequently detected using labeled antibodies (e.g. labeled sheep,goat, or mouse antibodies) that specifically bind to the anti-PRL-1.

5. ELISA

The present invention may also employ the use of immunoassays such as anenzyme linked immunosorbent assay (ELISA) in assessing the activity orexpression of PRL-1 in a cancer or tumor cell. An ELISA generallyinvolves the steps of coating, incubating and binding, washing to removespecies that are non-specifically bound, and detecting the bound immunecomplexes. This technique is well known in the art, for example see U.S.Pat. No. 4,367,110 and Harlow and Lane, 1988.

In an ELISA assay, a PRL-1 protein sample may be immobilized onto aselected surface, preferably a surface exhibiting a protein affinitysuch as the wells of a polystyrene microtiter plate. After washing toremove incompletely adsorbed material, it is desirable to bind or coatthe assay plate wells with a nonspecific protein that is known to beantigenically neutral with regard to the test antisera such as bovineserum albumin (BSA), casein or solutions of milk powder. This allows forblocking of nonspecific adsorption sites on the immobilizing surface andthus reduces the background caused by nonspecific binding of antiseraonto the surface.

After binding of the antigenic material to the well, coating with anon-reactive material to reduce background, and washing to removeunbound material, the immobilizing surface is contacted with theantisera or clinical or biological extract to be tested in a mannerconducive to immune complex (antigen/antibody) formation. Suchconditions preferably include diluting the antisera with diluents suchas BSA, bovine gamma globulin (BGG) and phosphate buffered saline(PBS)/Tween. These added agents also tend to assist in the reduction ofnonspecific background. The layered antisera is then allowed to incubatefor from 2 to 4 or more hours to allow effective binding, attemperatures preferably on the order of 25° C. to 37° C. (or overnightat 4° C.). Following incubation, the antisera-contacted surface iswashed so as to remove non-immunocomplexed material. A preferred washingprocedure includes washing with a solution such as PBS/Tween, or boratebuffer.

Following formation of specific immunocomplexes between the test sampleand the bound antigen and subsequent washing the occurrence and evenamount of immunocomplex formation may be determined by subjecting thesample to a second antibody having specificity for the first. To providea detecting means, the second antibody preferably has an associatedenzyme that generates a color development upon incubating with anappropriate chromogenic substrate. Thus, for example, one will desire tocontact and incubate the antisera-bound surface with a urease orperoxidase-conjugated anti-human IgG for a period of time and underconditions which favor the development of immunocomplex formation (e.g.,incubation for 2 hours at room temperature in a PBS-containing solutionsuch as PBS-Tween).

After incubation with the second enzyme-tagged antibody, and subsequentto washing to remove unbound material, the amount of label is quantifiedby incubation with a chromogenic substrate such as urea and bromocresolpurple or 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS)and H₂O₂, in the case of peroxidase as the enzyme label. Quantificationis then achieved by measuring the degree of color generation, e.g.,using a visible spectra spectrophotometer. The use of labels forimmunoassays are described in U.S. Pat. Nos. 5,310,687, 5,238,808 and5,221,605.

Other immunodetection methods that may be contemplated in the presentinvention include radioimmunoassay (RIA), immunoradiometric assay,fluoroimmunoassay, chemiluminescent assay, bioluminescent assay. Thesemethods are well known to those of ordinary skill and have beendescribed in Doolittle et al. (1999); Gulbis et al. (1993); De Jager etal. (1993); and Nakamura et al (1987), each incorporated herein byreference.

6. Tissue Microarray Immunohistochemistry

Tissue microarray immunohistochemistry is a recently developed techniquethat enables the simultaneous examination of multiple tissues sectionsconcurrently as compared to the more conventional technique of onesection at a time. This technique is used for high throughput molecularprofiling of tumor specimen (Kononen et al., 1998). More specifically,the present invention utilizes a pancreatic tumor tissue microarraycontaining different adenocarcinoma tissue samples, each of which havingtwo representative 1.5 mm disks from the different areas of the sameparaffin-embedded section. These pancreatic tissue microarrays may beused to verify the overexpression of other genes manifested in the cDNAmicroarray.

7. Determination of Circulating Cancer Cells

With the advent of enrichment techniques, detection of circulatingcancer cells can be used for the early detection of cancer recurrenceafter treatment of a primary tumor, early diagnosis of metastasis anduse in selection and monitoring of treatment strategies for varioustumors (Martin et al., 1998; Wang et al., 2000; Hu et al., 2003).Anti-PRL antibodies of the present invention may be used in conjunctionwith cancer cell enrichment techniques in the detection of circulatingpancreatic cancer cells. One suitable cell enrichment methodology is themagnetic-activated cell separation system as distributed by MiltenyiBiotec Inc. (Auburn, Calif.). This immunomagnetic method usesmagnetically labeled anti-cytokeratin 8 antibodies to separatecirculating cancer cells from other circulating cell types (see Martinet al., 1998; Hu et al., 2003). Pancreatic cancer cells expresscytokeratin 8 (Rafie et al., 1992; Ditzel et al., 1997; Luttges et al.,1998). For example, blood samples (20-40 ml) are collected, treated withanticoagulant and stored for up to 23 hr. until further processing whenthey are spun down at 400 g for 35 minutes and the leukocyte-richinterphase cells are collected and permeabilized with PBS containing0.5% BSA and 0.1% saponin and then fixed with 37% formaldehyde. Afterwashing twice with PBS, 0.5% BSA, 0.5% saponin and 0.05% NaN₃, the cellsare resuspended in 600 μl PBS, 0.5% BSA, 0.5% saponin and 0.05% NaN₃,and 200 μl FcR blocking reagent (Miltenyi Biotech) is added and thecancer cells directly magnetically labeled by the addition of 200 μlCytokeratin Microbeads (Miltenyi Biotec, Auburn, Calif.) and incubatingthe cells for 45 min. at room temperature. The magnetically labeledcells are passed through a 30 μm filter and applied to a MACS MSenrichment column (Miltenyi Biotec), which is located within a magneticfield. Negative cells are washed of with PBS, 0.5% BSA, and 0.05% NaN₃,and then labeled cells are removed using the same buffer and the plungersupplied with the column after removal of the column from the magneticfield. Pancreatic cancer cells in this fraction can be detected byimmunohistochemistry or flow cytometry using suitably labeled anti-PRL-1antibodies. Alternatively, magnetically anti-PRL-1 antibodies may beused to enrich circulating pancreatic cancer cells.

An alternative enrichment technique is Circulating Cancer Cell Test(Cell Works Inc., Baltimore, Md.; see Wang et al., 2000). This procedureutilizes a double gradient sedimentation for the removal of most RBC andWBC as well as magnetic cell sorting for the additional removal of WBCbefore spreading the cancer cells onto a slide utilizing a cytospinapparatus. The fixed cells on the slide are then stained with a suitablyanti-PRL-1 antibody and positive cells are automatically scanned with anspectroscopic microscope system, first in low magnification, where thefluorescent digital image is captured at a resolution of 0.2 μm usingmultiple excitation/emission wavelengths, then at higher resolution forfurther analysis. The system has automatic adjustment of exposure, focusand other parameters required for proper image acquisition and analysisto identify cancer cells and markers on the basis of intensity and blobanalysis.

8. Whole Body Imaging

The present invention may further employ the use of whole body imagingtechniques to identify subjects who have or may be at risk of developingcancer. Such diagnostic methods may employ positron emission tomography(PET) scanning, electron beam tomography (EBT) scanning, and MRIscanning. Essential to these methods is the use of labeled targetingagents, such as antibodies, that colocalize with PRL-1 in a quantitativefashion.

D. Screening Methods for PRL-1 Activity or Expression

1. Screening for Inhibitors of PRL-1

The present invention further comprises methods for identifyinginhibitors of PRL-1 activity or expression. PRL-1 may be used as atarget in screening for compounds that inhibit, decrease ordown-regulate its expression or activity in cancer cells, such aspancreatic cancer cells. These assays may comprise random screening oflarge libraries of candidate substances. Alternatively, the assays maybe used to focus on particular classes of compounds selected with an eyetowards structural attributes that are believed to make them more likelyto inhibit the function of PRL-1. By function, it is meant that one mayassay for inhibition of expression of PRL-1 in cancer cells, increaseapoptosis, or inhibition of the ability of the PRL-1 enzyme to cleavephosphatases off of the substrate.

To identify a PRL-1 inhibitor, one generally will determine PRL-1activity or expression in the presence and absence of the candidatesubstance, wherein an inhibitor is defined as any substance thatdown-regulates, reduces, inhibits or decreases PRL-1 activity orexpression. For example, a method may generally comprise:

-   -   a) providing a cell;    -   b) contacting the cell with a candidate compound; and    -   c) assessing the effect of the candidate compound on PRL-1        expression or activity,        wherein a decrease in the amount of PRL-1 expression or        activity, as compared to the amount of PRL-1 expression or        activity in a similar cell not treated with the candidate        compound, indicates that the candidate compound has anti-cancer        activity.

Assays may be conducted in cell free systems, in isolated cells, or inorganisms including transgenic animals. It will, of course, beunderstood that all the screening methods of the present invention areuseful in themselves notwithstanding the fact that effective candidatesmay not be found. The invention provides methods for screening for suchcandidates, not solely methods of finding them.

a. Inhibitors

As used herein the term “candidate substance” or “candidate compound”refers to any molecule that may potentially inhibit the expression oractivity of PRL-1. A PRL-1 inhibitor, may be a compound that overallaffects an inhibition of PRL-1 activity, which may be accomplished byinhibiting PRL-1 expression, translocation or transport, function,expression, post-translational modification, location, or more directlyby preventing its activity, such as by binding PRL-1. Any compound ormolecule described in the methods and compositions herein may be aninhibitor of PRL-1 activity or expression.

The candidate substance may be a protein or fragment thereof, a smallmolecule, or even a nucleic acid molecule. It may prove to be the casethat the most useful pharmacological compounds will be compounds thatare structurally related to PRL-1 or other protein tyrosinephosphatases, or that binds PRL-1. Using lead compounds to help developimproved compounds is known as “rational drug design” and include notonly comparisons with known inhibitors, but predictions relating to thestructure of target molecules.

Candidate compounds or inhibitors of the present invention will likelyfunction inhibit decrease or down-regulate the expression or activity ofPRL-1 in a cancer cell such as a pancreatic cancer cell. Such candidatecompounds may be inhibitors or regulators of protein tyrosinephosphatases; may have the ability to remove a phosphate from proteinsor peptides containing phosphotyrosine; or may likely be involved incontrolling cellular proliferation in a cancer or tumor cell, such aspancreatic cancer cells. These candidate compounds may be antisensemolecules, ribozymes, interfering RNAs, antibodies (including singlechain antibodies), or organopharmaceuticals, but are not limited tosuch.

b. Rational Drug Design

The present invention also provides methods for developing drugs thatinhibit PRL-1 activity or expression that may be used to treat a cancer,such as pancreatic cancer. One such method involves the prediction ofthe three dimensional structure of a validated protein tyrosinephosphatase target using molecular modeling and computer stimulations.The resulting structure may then be used in docking studies to identifypotential small molecule inhibitors that bind in the enzyme's activesite with favorable binding energies. Inhibitors identified may then betested in biochemical assays to further identify PRL-1 drug target forpancreatic cancer treatment.

Rational drug design is therefore used to produce structural analogs ofphosphorylated substrates for PRL-1. By creating such analogs, it ispossible to fashion drugs which are more active or stable than thenatural molecules, which have different susceptibility to alteration orwhich may affect the function of various other molecules. In oneapproach, one would generate a three-dimensional structure for the PRL-1targets of the invention or a fragment thereof. This could beaccomplished by X-ray crystallography, computer modeling or by acombination of both approaches.

It also is possible to use antibodies to ascertain the structure of atarget compound inhibitor. In principle, this approach yields aphannacore upon which subsequent drug design can be based. It ispossible to bypass protein crystallography altogether by generatinganti-idiotypic antibodies to a functional, pharmacologically activeantibody. As a mirror image of a mirror image, the binding site ofanti-idiotype would be expected to be an analog of the original antigen.The anti-idiotype could then be used to identify and isolate peptidesfrom banks of chemically- or biologically-produced peptides. Selectedpeptides would then serve as the pharmacore. Anti-idiotypes may begenerated using the methods described herein for producing antibodies,using an antibody as the antigen.

On the other hand, one may simply acquire, from various commercialsources, small molecule libraries that are believed to meet the basiccriteria for useful drugs in an effort to “brute force” theidentification of useful compounds. Screening of such libraries,including combinatorially generated libraries (e.g., peptide libraries),is a rapid and efficient way to screen large number of related (andunrelated) compounds for activity. Combinatorial approaches also lendthemselves to rapid evolution of potential drugs by the creation ofsecond, third and fourth generation compounds modeled of active, butotherwise undesirable compounds.

Candidate compounds may include fragments or parts ofnaturally-occurring compounds, or may be found as active combinations ofknown compounds, which are otherwise inactive. It is proposed thatcompounds isolated from natural sources, such as animals, bacteria,fungi, plant sources, including leaves and bark, and marine samples maybe assayed as candidates for the presence of potentially usefulpharmaceutical agents. It will be understood that the pharmaceuticalagents to be screened could also be derived or synthesized from chemicalcompositions or man-made compounds. Thus, it is understood that thecandidate substance identified by the present invention may be peptide,polypeptide, polynucleotide, small molecule inhibitors or any othercompounds that may be designed through rational drug design startingfrom known inhibitors or stimulators.

Other suitable compounds include antisense molecules, ribozymes, andantibodies (including single chain antibodies), each of which would bespecific for the target molecule. Such compounds are described ingreater detail elsewhere in this document. For example, an antisensemolecule that bound to a translational or transcriptional start site, orsplice junctions, would be ideal candidate inhibitors.

In addition to the inhibiting compounds initially identified, theinventors also contemplate that other sterically similar compounds maybe formulated to mimic the key portions of the structure of theinhibitors. Such compounds, which may include peptidomimetics of peptideinhibitors, may be used in the same manner as the initial inhibitors.

An inhibitor according to the present invention may be one which exertsits inhibitory or activating effect upstream, downstream or directly onPRL-1 or other related phosphates of this gene family. Regardless of thetype of inhibitor identified by the present screening methods, theeffect of the inhibition by such a compound results in the regulation inPRL-1 activity or expression as compared to that observed in the absenceof the added candidate substance.

The term “drug” is intended to refer to a chemical entity, whether inthe solid, liquid, or gaseous phase which is capable of providing adesired therapeutic effect when administered to a subject. The term“drug” should be read to include synthetic compounds, natural productsand macromolecular entities such as polypeptides, polynucleotides, orlipids and also small entities such as neurotransmitters, ligands,hormones or elemental compounds. The term “drug” is meant to refer tothat compound whether it is in a crude mixture or purified and isolated.

c. Bioisosterism

The present invention also contemplates the application ofbioisosterism, the concept of isosterism to modify biological activityof a lead compound, in developing drugs that cacn inhibit PRL-1 activityor expression that may be used as therapeutic agents. As discussedabove, a lead compound with a desired pharmacological activity may haveassociated with it undesirable side effects, characteristics that limitits bioavailability, or structural features which adversely influenceits metabolism and excretion from the body. Bioisosterism represents oneapproach used in the art for the rational modification of lead compoundsinto safer and more clinically effective agents (Patani and LaVoie,1996). The ability of a group of bioisosteres to elicit similarbiological activity has been attributed to common physicochemicalproperties such as electro-negativity, steric size, and lipophilicity.Bioisosteric replacements of functional groups based on theunderstanding of the pharmacophore and the physicochemical properties ofthe bioisosteres have enhanced the potential for the successfuldevelopment of new clinical agents. A critical component forbioisosterism is that bioisosteres affect the same pharmacologicaltarget as agonists or antagonists and, thereby, have biologicalproperties which are related to each other.

Bioisosteres are classified as either classical or nonclassical.Classical bioisosteres have been traditionally divided into severaldistinct categories: (a) monovalent atoms or groups; (b) divalent atomsor groups; (c) trivalent atoms or groups; (d) tetrasubstituted atoms;and (e) ring equivalents. Nonclassical bioisosteres can be divided into(a) rings vs noncyclic structures; and (b) exchangeable groups.Nonclassical isosteres differ from that of the classical bioisosteres inthat they do not obey the steric and electronic definition of classicalisosteres. A notable characteristic of nonclassical bioisosteres is thatthey do not have the same number of atoms as the substituent or moietyfor which they are used as a replacement.

In the present invention the application of bioisosterism has beenemployed in developing agents that can inhibit PRL-1 activity orexpression. For example, the pharmacophore of the a lead compound,UA668394, may be exploited using the concept of bioisosterism to developthe analogs UA668394-1 and UA668394-2 as provided below:

wherein R¹ is hydrogen, halogen, thiol, trifluoromethyl, or hydroxyl andR² is a hydrogen, halogen, thiol, hydroxyl or trifluoromethyl,

wherein R³ and R⁵ are independently halogen, thiol, hydroxy ortrifluoromethyl, and R⁴ is hydroxyl, halogen, thiol, trifluoromethyl,CH₂OH, NHCONH₂, NHSO₂CH₃, or NHCN,

wherein R³ and R⁵ are independently halogen, thiol, hydroxyl ortrifluoromethyl, and R⁴ is hydroxyl, halogen, thiol, trifluoromethyl,CH₂OH, NHCONH₂, NHSO₂CH₃, or NHCN.

2. Phosphatase Assays

a. Tyrosine Phosphatase Assay

Assays that measure the removal of phosphates from proteins or peptidescontaining phosphotyrosine may also be employed in the presentinvention. One method of screening for drug targets would involvemeasuring inhibition of PRL-1-mediated tyrosine dephosphorylation. Thisassay detects the amount of free phosphatase generated in a reaction bymeasuring the absorbance of a molybdate:malachite green:phosphatecomplex. This assay detects the activity of protein tyrosinephosphatases. Such assays or systems are commercially available fromsuppliers such as Promega (Madison, Wis.) or Applied Biosystems (FosterCity, Calif.).

b. DiFMUP Assay

Another assay employed in the present invention is an improved methodfor measuring protein phosphatases for high-throughput screeninginvolving 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP). DiFMUPcan assay both acid and alkaline phosphatase activity. The hydrolysisproduct of DiFMUP to DiF4MU exhibits both a lower pka (4.9 versus 7.8)and a higher fluorescence quantum yield (0.89 versus 0.63) than thehydrolysis product of MUP. The lower pka of its hydrolysis product makesDiFMUP a sensitive substrate for acid phosphatases, which is notpossible with MUP because its fluorescence must be measured at alkalinepH. Furthermore, with its high quantum yield, DiFMUP increases thesensitivity of both acid and alkaline phosphatase measurements. As withfluorinated fluorescein derivatives (i.e., Oregon Green dyes)fluorination reduces the susceptibility of the methylumbelliferonefluorophore to photobleaching effects without significantly affectingthe extinction coefficient or excitation/emission maxima. DiFMUP enablesthe quantitation of as little as 1.0 pg/ml alkaline phosphatase.

For example, in the present invention, a bacterial expression system maybe employed (i.e., pProEx vector) from which recombinant His-taggedPRl-1 protein may be obtained and purified using a column (i.e., anickel column). The PRl-1 enzymatic activity in the presence of a drugcompound of interest and in combination with DiFMUP substrate may beincubated (about 1 h) and the dephosphorylated substrate detected.

3. In Vitro Assays

A quick, inexpensive and easy assay to run is an in vitro assay. Suchassays generally use isolated molecules, and can be run quickly and inlarge numbers, thereby increasing the amount of information obtainablein a short period of time. A variety of vessels may be used to run theassays, including test tubes, plates, dishes and other surfaces such asdipsticks or beads.

One example of a cell-free assay is a binding assay. While not directlyaddressing function, the ability of a compound to bind to a targetmolecule such as PRL-1 in a specific fashion is strong evidence of arelated biological effect, which can be assessed in follow on screens.For example, binding of a molecule to PRL-1 may, in and of itself, beinhibitory, due to steric, allosteric or charge-charge interactions. ThePRL-1 may be either free in solution, fixed to a support, expressed inor on the surface of a cell. Either the PRL-1 or the compound may belabeled, thereby permitting measuring of the binding. Competitivebinding formats can be performed in which one of the agents is labeled,and one may measure the amount of free label versus bound label todetermine the effect on binding.

A technique for high throughput screening of compounds is described inWO 84/03564. Large numbers of small peptide test compounds aresynthesized on a solid substrate, such as plastic pins or some othersurface. Bound polypeptide is detected by various methods.

4. In Cyto Assays

The present invention also contemplates the screening of compounds fortheir ability to inhibit PRL-1 in cells. Various cell lines can beutilized for such screening assays, including cells specificallyengineered for this purpose. The present invention particularlycontemplates the use of pancreatic cancer cells, which express a higherlevel of PRL-1 activity, and thus may provide an easier baseline formeasurement. Depending on the assay, culture may be required. The cellis examined using any of a number of different physiologic assays.Alternatively, molecular analysis may be performed, for example, lookingat protein expression, mRNA expression (including differential displayof whole cell or polyA RNA) and others by methods as described hereinand that are well known to those of skill in the art.

5. In Vivo Assays

In vivo assays involve the use of various animal models, includingtransgenic animals that have been engineered to have specific defectssuch as PRL-1 overexpression, or that carry markers that can be used tomeasure the ability of a candidate substance to reach and effectdifferent cells within the organism. Due to their size, ease ofhandling, and information on their physiology and genetic make-up, miceare a preferred embodiment, especially for transgenics. However, otheranimals are suitable as well, including rats, rabbits, hamsters, guineapigs, gerbils, woodchucks, cats, dogs, sheep, goats, pigs, cows, horsesand monkeys (including chimps, gibbons and baboons). Assays forinhibitors may be conducted using an animal model derived from any ofthese species.

In such assays, one or more candidate substances are administered to ananimal, and the ability of the candidate substance(s) to alter one ormore characteristics, as compared to a similar animal not treated withthe candidate substance(s), identifies an inhibitor. The characteristicsmay be any of those discussed above with regard to PRL-1 expression orfunction, or it may be broader in the sense of “treating” the conditionpresent in the animal.

Treatment of these animals with test compounds will involve theadministration of the compound, in an appropriate form, to the animal.Administration will be by any route that could be utilized for clinicalor non-clinical purposes, including but not limited to oral, nasal,buccal, or even topical. Alternatively, administration may be byintratracheal instillation, bronchial instillation, intradermal,subcutaneous, intramuscular, intraperitoneal or intravenous injection.Specifically contemplated routes are systemic intravenous injection,regional administration via blood or lymph supply, or directly to anaffected site.

Determining the effectiveness of a compound in vivo may involvemeasuring toxicity and dose response can be performed in animals in amore meaningful fashion than in in vitro or in cyto assays.

E. Cancer Treatment

The present invention embodies a method of treating cancer such aspancreatic cancer, by the delivery of a PRL-1 inhibitor to a subjecthaving a cancer. Examples of cancers contemplated for treatment includeleukemia, ovarian cancer, breast cancer, lung cancer, colon cancer,liver cancer, prostate cancer, testicular cancer, stomach cancer, braincancer, bladder cancer, head and neck cancer, melanoma, and any othercancer that may be treated by inhibiting or decreasing the activity ofPRL-1 activity.

1. PRL-1 Inhibitors

a. Antisense

Antisense methodology takes advantage of the fact that nucleic acidstend to pair with “complementary” sequences. By complementary, it ismeant that polynucleotides are those which are capable of base-pairingaccording to the standard Watson-Crick complementarity rules. That is,the larger purines will base pair with the smaller pyrimidines to formcombinations of guanine paired with cytosine (G:C) and adenine pairedwith either thymine (A:T) in the case of DNA, or adenine paired withuracil (A:U) in the case of RNA. Inclusion of less common bases such asinosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others inhybridizing sequences does not interfere with pairing.

Targeting double-stranded (ds) DNA with polynucleotides leads totriple-helix formation; targeting RNA will lead to double-helixformation. Antisense polynucleotides, when introduced into a targetcell, specifically bind to their target polynucleotide and interferewith transcription, RNA processing, transport, translation and/orstability. Antisense RNA constructs, or DNA encoding such antisenseRNAs, may be employed to inhibit gene transcription or translation orboth within a host cell, either in vitro or in vivo, such as within ahost animal, including a human subject.

Antisense constructs may be designed to bind to the promoter and othercontrol regions, exons, introns or even exon-intron boundaries of agene. It is contemplated that the most effective antisense constructsmay include regions complementary to intron/exon splice junctions. Thus,antisense constructs with complementarity to regions within 50-200 basesof an intron-exon splice junction may be used. It has been observed thatsome exon sequences can be included in the construct without seriouslyaffecting the target selectivity thereof. The amount of exonic materialincluded will vary depending on the particular exon and intron sequencesused. One can readily test whether too much exon DNA is included simplyby testing the constructs in vitro to determine whether normal cellularfunction is affected or whether the expression of related genes havingcomplementary sequences is affected.

As stated above, “complementary” or “antisense” means polynucleotidesequences that are substantially complementary over their entire lengthand have very few base mismatches. For example, sequences of fifteenbases in length may be termed complementary when they have complementarynucleotides at thirteen or fourteen positions. Naturally, sequenceswhich are completely complementary will be sequences which are entirelycomplementary throughout their entire length and have no basemismatches. Other sequences with lower degrees of homology also arecontemplated. For example, an antisense construct which has limitedregions of high homology, but also contains a non-homologous region(e.g., ribozyme) could be designed. These molecules, though having lessthan 50% homology, would bind to target sequences under appropriateconditions.

It may be advantageous to combine portions of genomic DNA with cDNA orsynthetic sequences to generate specific constructs. For example, wherean intron is desired in the ultimate construct, a genomic clone willneed to be used. The cDNA or a synthesized polynucleotide may providemore convenient restriction sites for the remaining portion of theconstruct and, therefore, would be used for the rest of the sequence.

b. Ribozymes

The present invention also contemplates the use of PRL-1-specificribozymes to down-regulate or inhibit PRL-1 expression. Ribozymes areRNA-protein complexes that cleave nucleic acids in a site-specificfashion. Ribozymes have specific catalytic domains that possessendonuclease activity (Kim and Cech, 1987; Forster and Symons, 1987).For example, a large number of ribozymes accelerate phosphoestertransfer reactions with a high degree of specificity, often cleavingonly one of several phosphoesters in an oligonucleotide substrate (Cechet. al., 1981; Michel and Westhof, 1990; Reinhold-Hurek and Shub, 1992).This specificity has been attributed to the requirement that thesubstrate bind via specific base-pairing interactions to the internalguide sequence (“IGS”) of the ribozyme prior to chemical reaction.

Ribozyme catalysis has primarily been observed as part of sequencespecific cleavage/ligation reactions involving nucleic acids (Joyce,1989; Cech et. al., 1981). For example, U.S. Pat. No. 5,354,855 reportsthat certain ribozymes can act as endonucleases with a sequencespecificity greater than that of known ribonucleases and approachingthat of the DNA restriction enzymes. Thus, sequence-specificribozyme-mediated inhibition of gene expression (Scanlon et al., 1991;Sarver et. al., 1990; Sioud et. al., 1992) is particularly suited totherapeutic applications of the present invention. It has been reportedthat ribozymes elicited genetic changes in some cell lines to which theywere applied; the altered genes included the oncogenes H-ras, c-fos andgenes of HIV. Most of this work involved the modification of a targetmRNA, based on a specific mutant codon that is cleaved by a specificribozyme. In light of the information included herein and the knowledgeof one of ordinary skill in the art, the preparation and use ofadditional ribozymes that are specifically targeted to a given gene willnow be straightforward.

Several different ribozyme motifs have been described with RNA cleavageactivity (reviewed in Symons, 1992). Examples that would be expected tofunction equivalently for the down-regulation or inhibition of PRl-1include sequences from the Group I self splicing introns includingtobacco ringspot virus (Prody et. al., 1986), avocado sunblotch viroid(Palukaitis et. al., 1979), and Lucerne transient streak virus (Forsterand Symons, 1987). Sequences from these and related viruses are referredto as hammerhead ribozymes based on a predicted folded secondarystructure.

Other suitable ribozymes include sequences from RNase P with RNAcleavage activity (Yuan et al., 1992; Yuan and Altman, 1994), hairpinribozyme structures (Berzal-Herranz et al., 1992; Chowrira et al., 1993)and hepatitis virus based ribozymes (Perrotta and Been, 1992). Thegeneral design and optimization of ribozyme directed RNA cleavageactivity has been discussed in detail (Haseloff and Gerlach, 1988;Symons, 1992; Chowrira, et al., 1994; and Thompson, et al., 1995).

The other variable on ribozyme design is the selection of a cleavagesite on a given target RNA. Ribozymes are targeted to a given sequenceby virtue of annealing to a site by complimentary base pairinteractions. Two stretches of homology are required for this targeting.These stretches of homologous sequences flank the catalytic ribozymestructure defined above. Each stretch of homologous sequence can vary inlength from 7 to 15 nucleotides. The only requirement for defining thehomologous sequences is that, on the target RNA, they are separated by aspecific sequence which is the cleavage site. For hammerhead ribozymes,the cleavage site is a dinucleotide sequence on the target RNA, uracil(U) followed by either an adenine, cytosine or uracil (A,C or U;Perriman, et al., 1992; Thompson, et al., 1995). The frequency of thisdinucleotide occurring in any given RNA is statistically 3 out of 16.Therefore, for a given target mRNA of 1000 bases, 187 dinucleotidecleavage sites are statistically possible.

Designing and testing ribozymes for efficient cleavage of a target RNAis a process well known to those skilled in the art. Examples ofscientific methods for designing and testing ribozymes are described byChowrira et al. (1994) and Lieber and Strauss (1995), each incorporatedby reference. The identification of operative and preferred sequencesfor use in PRL-1-targeted ribozymes is simply a matter of preparing andtesting a given sequence, and is a routinely practiced “screening”method known to those of skill in the art.

C. RNA Interference (RNAi)

RNA interference (also referred to as “RNA-mediated interference” orRNAi) is a mechanism by which gene expression can be reduced oreliminated. Double stranded RNA (dsRNA) has been observed to mediate thereduction, which is a multi-step process. dsRNA activatespost-transcriptional gene expression surveillance mechanisms that appearto function to defend cells from virus infection and transposonactivity. (Fire et al., 1998; Grishok et al., 2000; Ketting et al.,1999; Lin et al., 1999; Montgomery et al., 1998; Sharp et al., 2000;Tabara et al., 1999). Activation of these mechanisms targets mature,dsRNA-complementary mRNA for destruction. RNAi offers major experimentaladvantages for study of gene function. These advantages include a veryhigh specificity, ease of movement across cell membranes, and prolongeddown-regulation of the targeted gene. (Fire et al., 1998; Grishok etal., 2000; Ketting et al., 1999; Lin et al., 1999; Montgomery et al.,1998; Sharp, 1999; Sharp et al., 2000; Tabara et al., 1999). Moreover,dsRNA has been shown to silence genes in a wide range of systems,including plants, protozoans, fungi, C. elegans, Trypanasoma,Drosophila, and mammals (Grishok et al., 2000; Sharp, 1999; Sharp etal., 2000; Elbashir et al., 2001). It is generally accepted that RNAiacts post-transcriptionally, targeting RNA transcripts for degradation.It appears that both nuclear and cytoplasmic RNA can be targeted.(Bosher et al., 2000).

siRNAs must be designed so that they are specific and effective insuppressing the expression of the genes of interest. Methods ofselecting the target sequences, i.e. those sequences present in the geneor genes of interest to which the siRNAs will guide the degradativemachinery, are directed to avoiding sequences that may interfere withthe siRNA's guide function while including sequences that are specificto the gene or genes. Typically, siRNA target sequences of about 21 to23 nucleotides in length are most effective. This length reflects thelengths of digestion products resulting from the processing of muchlonger RNAs as described above. (Montgomery et al., 1998).

The making of siRNAs has been mainly through direct chemical synthesis;through processing of longer, double stranded RNAs through exposure toDrosophila embryo lysates; or through an in vitro system derived from S2cells. Use of cell lysates or in vitro processing may further involvethe subsequent isolation of the short, 21-23 nucleotide siRNAs from thelysate, etc., making the process somewhat cumbersome and expensive.Chemical synthesis proceeds by making two single stranded RNA-oligomersfollowed by the annealing of the two single stranded oligomers into adouble stranded RNA. Methods of chemical synthesis are diverse.Non-limiting examples are provided in U.S. Pat. Nos. 5,889,136;4,415,732; 4,458,066, expressly incorporated herein by reference, and inWincott et. al. (1995).

Several further modifications to siRNA sequences have been suggested inorder to alter their stability or improve their effectiveness. It issuggested that synthetic complementary 21-mer RNAs having di-nucleotideoverhangs (i.e., 19 complementary nucleotides +3′ non-complementaiydimers) may provide the greatest level of suppression. These protocolsprimarily use a sequence of two (2′-deoxy) thymidine nucleotides as thedi-nucleotide overhangs. These dinucleotide overhangs are often writtenas dTdT to distinguish them from the typical nucleotides incorporatedinto RNA. The literature has indicated that the use of dT overhangs isprimarily motivated by the need to reduce the cost of the chemicallysynthesized RNAs. It is also suggested that the dTdT overhangs might bemore stable than UU overhangs, though the data available shows only aslight (<20%) improvement of the dTdT overhang compared to an siRNA witha UU overhang.

Chemically synthesized siRNAs are found to work optimally when they arein cell culture at concentrations of 25-100 nM. This had beendemonstrated by Elbashir et. al. wherein concentrations of about 100 nMachieved effective suppression of expression in mammalian cells. siRNAshave been most effective in mammalian cell culture at about 100 nM. Inseveral instances, however, lower concentrations of chemicallysynthesized siRNA have been used (Caplen et. al., 2000; Elbashir et.al., 2001).

WO 99/32619 and WO 01/68836 suggest that RNA for use in siRNA may bechemically or enzymatically synthesized. Both of these texts areincorporated herein in their entirety by reference. The enzymaticsynthesis contemplated in these references is by a cellular RNApolymerase or a bacteriophage RNA polymerase (e.g. T3, T7, SP6) via theuse and production of an expression construct as is known in the art.For example, see U.S. Pat. No. 5,795,715. The contemplated constructsprovide templates that produce RNAs that contain nucleotide sequencesidentical to a portion of the target gene. The length of identicalsequences provided by these references is at least 25 bases, and may beas many as 400 or more bases in length. An important aspect of thisreference is that the authors contemplate digesting longer dsRNAs to21-25mer lengths with the endogenous nuclease complex that converts longdsRNAs to siRNAs in vivo. They do not describe or present data forsynthesizing and using in vitro transcribed 21-25mer dsRNAs. Nodistinction is made between the expected properties of chemical orenzymatically synthesized dsRNA in its use in RNA interference.

Similarly, WO 00/44914, incorporated herein by reference, suggests thatsingle strands of RNA can be produced enzymatically or by partial/totalorganic synthesis. Preferably, single stranded RNA is enzymaticallysynthesized from the PCR™ products of a DNA template, preferably acloned cDNA template and the RNA product is a complete transcript of thecDNA, which may comprise hundreds of nucleotides. WO 01/36646,incorporated herein by reference, places no limitation upon the mannerin which the siRNA is synthesized, providing that the RNA may besynthesized in vitro or in vivo, using manual and/or automatedprocedures. This reference also provides that in vitro synthesis may bechemical or enzymatic, for example using cloned RNA polymerase (e.g.,T3, T7, SP6) for transcription of the endogenous DNA (or cDNA) template,or a mixture of both. Again, no distinction in the desirable propertiesfor use in RNA interference is made between chemically or enzymaticallysynthesized siRNA.

U.S. Pat. No. 5,795,715 reports the simultaneous transcription of twocomplementary DNA sequence strands in a single reaction mixture, whereinthe two transcripts are immediately hybridized. The templates used arepreferably of between 40 and 100 base pairs, and which is equipped ateach end with a promoter sequence. The templates are preferably attachedto a solid surface. After transcription with RNA polymerase, theresulting dsRNA fragments may be used for detecting and/or assayingnucleic acid target sequences.

2. Pharmaceutical Compositions and Routes of Administration

Pharmaceutical compositions of the present invention compriseadministering an effective amount of one or more inhibitors that inhibitor down-regulate the PRL-1 activity (and/or an additional agent)dissolved or dispersed in a pharmaceutically acceptable carrier to asubject. The phrases “pharmaceutical or pharmacologically acceptable”refers to molecular entities and compositions that do not produce anadverse, allergic or other untoward reaction when administered to ananimal, such as, for example, a human, as appropriate. The preparationof a pharmaceutical composition that contains at least one PRL-1inhibitor or additional active ingredient will be known to those ofskill in the art in light of the present disclosure, and as exemplifiedby Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference. Moreover, for animal (e.g.,human) administration, it will be understood that preparations shouldmeet sterility, pyrogenicity, general safety and purity standards asrequired by FDA Office of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for. example, Remington's PharmaceuticalSciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329,incorporated herein by reference). Except insofar as any conventionalcarrier is incompatible with the active ingredient, its use in thetherapeutic or pharmaceutical compositions is contemplated.

A pharmaceutical composition of the present invention may comprisedifferent types of carriers depending on whether it is to beadministered in solid, liquid or aerosol form, and whether it needs tobe sterile for such routes of administration as injection. Apharmaceutical composition of the present invention can be administeredintravenously, intradermally, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostaticaly,intrapleurally, intratracheally, intranasally, intravitreally,intravaginally, intrarectally, topically, intratumorally,intramuscularly, intraperitoneally, subcutaneously, subconjunctival,intravesicularlly, mucosally, intrapericardially, intraumbilically,intraocularally, orally, topically, locally, inhalation (e.g., aerosolinhalation), injection, infusion, continuous infusion, localizedperfusion bathing target cells directly, via a catheter, via a lavage,in cremes, in lipid compositions (e.g., liposomes), or by other methodor any combination of the foregoing as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, incorporated herein by reference).

The actual dosage amount of a composition of the present inventionadministered to a subject can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. The number of doses and the period of time over whichthe dose may be given may vary. The practitioner responsible foradministration will, in any event, determine the concentration of activeingredient(s) in a composition and appropriate dose(s), as well as thelength of time for administration for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0. 1% of an active compound. In otherembodiments, the active compound may comprise between about 2% to about75% of the weight of the unit, or between about 25% to about 60%, forexample, and any range derivable therein. In other non-limitingexamples, a dose may also comprise from about 1 microgram/kg/bodyweight, about 5 microgram/kg/body weight, about 10 microgram/kg/bodyweight, about 50 microgram/kg/body weight, about 100 microgram/kg/bodyweight, about 200 microgram/kg/body weight, about 350 microgram/kg/bodyweight, about 500 microgram/kg/body weight, about 1 milligram/kg/bodyweight, about 5 milligram/kg/body weight, about 10 milligram/kg/bodyweight, about 50 milligram/kg/body weight, about 100 milligram/kg/bodyweight, about 200 milligram/kglbody weight, about 350 milligram/kg/bodyweight, about 500 milligram/kg/body weight, to about 1000 mg/kg/bodyweight or more per administration, and any range derivable therein. Innon-limiting examples of a derivable range from the numbers listedherein, a range of about 5 mg/kg/body weight to about 100 mg/kg/bodyweight, about 5 microgram/kg/body weight to about 500 milligram/kg/bodyweight, etc., can be administered, based on the numbers described above.

In any case, the composition may comprise various antioxidants to retardoxidation of one or more component. Additionally, the prevention of theaction of microorganisms can be brought about by preservatives such asvarious antibacterial and antifungal agents, including but not limitedto parabens (e.g., methylparabens, propylparabens), chlorobutanol,phenol, sorbic acid, thimerosal or combinations thereof

A PRL-1 inhibitor(s) of the present invention may be formulated into acomposition in a free base, neutral or salt form. Pharmaceuticallyacceptable salts, include the acid addition salts, e.g., those formedwith the free amino groups of a proteinaceous composition, or which areformed with inorganic acids such as for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric ormandelic acid. Salts formed with the free carboxyl groups can also bederived from inorganic bases such as for example, sodium, potassium,ammonium, calcium or ferric hydroxides; or such organic bases asisopropylamine, trimethylamine, histidine or procaine.

In embodiments where the composition is in a liquid form, a carrier canbe a solvent or dispersion medium comprising but not limited to, water,ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethyleneglycol, etc), lipids (e.g., triglycerides, vegetable oils, liposomes)and combinations thereof. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin; by the maintenanceof the required particle size by dispersion in carriers such as, forexample liquid polyol or lipids; by the use of surfactants such as, forexample hydroxypropylcellulose; or combinations thereof such methods. Inmany cases, it will be preferable to include isotonic agents, such as,for example, sugars, sodium chloride or combinations thereof.

In certain aspects of the invention, the PRL-1 inhibitors are preparedfor administration by such routes as oral ingestion. In theseembodiments, the solid composition may comprise, for example, solutions,suspensions, emulsions, tablets, pills, capsules (e.g., hard or softshelled gelatin capsules), sustained release formulations, buccalcompositions, troches, elixirs, suspensions, syrups, wafers, orcombinations thereof. Oral compositions may be incorporated directlywith the food of the diet. Preferred carriers for oral administrationcomprise inert diluents, assimilable edible carriers or combinationsthereof. In other aspects of the invention, the oral composition may beprepared as a syrup or elixir. A syrup or elixir, and may comprise, forexample, at least one active agent, a sweetening agent, a preservative,a flavoring agent, a dye, a preservative, or combinations thereof.

In certain preferred embodiments an oral composition may comprise one ormore binders, excipients, disintegration agents, lubricants, flavoringagents, and combinations thereof. In certain embodiments, a compositionmay comprise one or more of the following: a binder, such as, forexample, gum tragacanth, acacia, cornstarch, gelatin or combinationsthereof; an excipient, such as, for example, dicalcium phosphate,mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate or combinations thereof; a disintegratingagent, such as, for example, corn starch, potato starch, alginic acid orcombinations thereof; a lubricant, such as, for example, magnesiumstearate; a sweetening agent, such as, for example, sucrose, lactose,saccharin or combinations thereof; a flavoring agent, such as, forexample peppermint, oil of wintergreen, cherry flavoring, orangeflavoring, etc.; or combinations thereof the foregoing. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, carriers such as a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both.

Additional formulations which are suitable for other modes ofadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum, vagina or urethra. After insertion, suppositoriessoften, melt or dissolve in the cavity fluids. In general, forsuppositories, traditional carriers may include, for example,polyalkylene glycols, triglycerides or combinations thereof. In certainembodiments, suppositories may be formed from mixtures containing, forexample, the active ingredient in the range of about 0.5% to about 10%,and preferably about 1% to about 2%.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and/or the otheringredients. In the case of sterile powders for the preparation ofsterile injectable solutions, suspensions or emulsion, the preferredmethods of preparation are vacuum-drying or freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessary andthe liquid diluent first rendered isotonic prior to injection withsufficient saline or glucose. The preparation of highly concentratedcompositions for direct injection is also contemplated, where the use ofDMSO as solvent is envisioned to result in extremely rapid penetration,delivering high concentrations of the active agents to a small area.

The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectablecomposition can be brought about by the use in the compositions ofagents delaying absorption, such as, for example, aluminum monostearate,gelatin or combinations thereof.

F. Combination Therapies with PRL-1 Inhibitor(s)

In order to increase the effectiveness of a cancer treatment with thecompositions of the present invention, such as a PRL-1 inhibitor, it maybe desirable to combine these compositions with other cancer therapyagents. For example, the treatment of a cancer may be implemented withtherapeutic agents of the present invention in conjunction with otheranti-cancer therapies. Thus, in the present invention, it iscontemplated that a PRL-1 inhibitor(s) may be used in conjunction with achemotherapeutic, a radiotherapeutic, an immunotherapeutic or otherbiological intervention, in addition to pro-apoptotic or cell cycleregulating agents or protein tyrosine phosphatase regulators.

This process may involve contacting the cell(s) with a PRL-1 inhibitorand a therapeutic agent at the same time or within a period of timewherein separate administration of the inhibitor and an agent to a cell,tissue or organism produces a desired therapeutic benefit. The terms“contacted” and “exposed,” when applied to a cell, tissue or organism,are used herein to describe the process by which a PRL-1 inhibitorand/or therapeutic agent are delivered to a target cell, tissue ororganism or are placed in direct juxtaposition with the target cell,tissue or organism. The cell, tissue or organism may be contacted (e.g.,by administration) with a single composition or pharmacologicalformulation that includes both a PRL-1 inhibitor and one or more agents,or by contacting the cell with two or more distinct compositions orformulations, wherein one composition includes a PRL-1 inhibitor and theother includes one or more agents.

1. Regimens

The PRL-1 inhibitor may precede, be concurrent with and/or follow theother agent(s) by intervals ranging from minutes to weeks. Inembodiments where the PRL-1 inhibitor and other agent(s) are appliedseparately to a cell, tissue or organism, one would generally ensurethat a significant period of time did not expire between the time ofeach delivery, such that the inhibitor and agent(s) would still be ableto exert an advantageously combined effect on the cell, tissue ororganism. For example, in such instances, it is contemplated that onemay contact the cell, tissue or organism with two, three, four or moremodalities substantially simultaneously (i.e., within less than about aminute) as the inhibitor. In other aspects, one or more agents may beadministered within of from substantially simultaneously, about 1minute, about 5 minutes, about 10 minutes, about 20 minutes about 30minutes, about 45 minutes, about 60 minutes, about 2 hours, or morehours, or about 1 day or more days, or about 4 weeks or more weeks, orabout 3 months or more months, or about one or more years, and any rangederivable therein, prior to and/or after administering the PRL-1inhibitor.

Various combinations of a PRL-1 inhibitor(s) and a cancer therapeuticmay be employed in the present invention, where a PRL-1 inhibitor is “A”and the secondary agent, such as a chemotherapeutic or radiotherapeuticagent, or any other cancer therapeutic agent is “B”: A/B/A B/A/B B/B/AA/A/B A/B/B B/A/A B/B/B/A B/B/A/ B A/A/B/B A/B/A/B A/B/B/A B/B/A/AB/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/BAdministration of a PRL-1 inhibitor of the present invention to apatient will follow general protocols for the administration of thatparticular secondary therapy, taking into account the toxicity, if any,of the PRL-1 inhibitor treatment. It is expected that the treatmentcycles would be repeated as necessary. The compositions employed ill thepresent invention may be administered once or more than once to asubject. It also is contemplated that various cancer therapies, such aschemotherapy, radiotherapy, as well as surgical intervention, may beapplied in combination with the described pancreatic cancer therapy.

2. Anti-Cancer Therapies

An “anti-cancer” agent as contemplated for use with the presentinvention would be capable of negatively affecting cancer in a subject,for example, by killing cancer cells, inducing apoptosis in cancercells, reducing the growth rate of cancer cells, reducing the incidenceor number of metastases, reducing tumor size, inhibiting tumor growth,reducing the blood supply to a tumor or cancer cells, promoting animmune response against cancer cells or a tumor, preventing orinhibiting the progression of cancer, or increasing the lifespan of asubject with cancer. Anti-cancer agents include biological agents(biotherapy), chemotherapy agents, and radiotherapy agents. Thecombination of chemotherapy with biological therapy is known asbiochemotherapy.

In the present invention a composition that inhibits PRL-1 activity andan anti-cancer agent would be provided in a combined amount effective tokill or inhibit proliferation of the cell. This process may involvecontacting the cells with the PRL-1 inhibitor and the agent(s) orfactor(s) at the same time. This may be achieved by contacting the cellwith a single composition or pharmacological formulation that includesboth the PRL-1 inhibitor and the other agent, or by contacting the cellwith two distinct compositions or formulations, at the same time,wherein one composition includes the PRL-1 inhibitor and the otherincludes the second agent(s).

a. Chemotherapy

It is also contemplated in the present invention a PRL-1 inhibitor(s)may be used in combination with chemotherapeutic agents. Suchchemotherapeutic agents may include, for example, cisplatin (CDDP),carboplatin, procarbazine, mechlorethamine, cyclophosphamide,camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea,dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin,mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptorbinding agents, taxol, gemcitabien, navelbine, famesyl-proteintransferase inhibitors, transplatinum, 5-fluorouracil, vincristine,vinblastine and methotrexate, Temazolomide (an aqueous form of DTIC), orany analog or derivative thereof. One example of a chemotherapuetucagent currently used to treat pancreatic cancer is gemcitaben. Otherstudies employ high doses of 5-Fluorouracil (5-FU) for treatment ofadvanced pancreatic cancer.

The PRL-1 inhibitors may also be used in combination with otherchemotherapeutic agents such as protein tyrosine kinase inhibitors. Suchinhibitors may suitably include imatinib or imatinib mesylate (STI-571,Gleevec™; Norvartis, Inc.,), OSI-774 (Tarceva™; OSI Pharmaceuticals,Inc.,), ZD-1839 (Iressa®); AstraZeneca, Inc.,), SU-101 (Sugen, Inc.,)and CP-701 (Cephalon, Inc.,).

b. Radiotherapy

Another therapy that may be used in conjunction with a PRL-1inhibitor(s) of the present invention to treat a cancer is radiotherapy.It is contemplated that radiotherapeutic factors that may be employed inthe present invention are factors that cause DNA damage and have beenused extensively, such as γ-rays, X-rays, and/or the directed deliveryof radioisotopes to tumor cells. Other forms of DNA damaging factors arealso contemplated such as microwaves and UV-irradiation. It is mostlikely that all of these factors effect a broad range of damage on DNA,on the precursors of DNA, on the replication and repair of DNA, and onthe assembly and maintenance of chromosomes. Dosage ranges for X-raysrange from daily doses of 50 to 200 roentgens for prolonged periods oftime (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosageranges for radioisotopes vary widely, and depend on the half-life of theisotope, the strength and type of radiation emitted, and the uptake bythe cancer or tumor cells.

c. Immunotherapy

The present invention also contemplates the use of immunotherapy inconjunction with a PRL-1 inhibitor(s). Immunotherapeutics, generally,rely on the use of immune effector cells and molecules to target anddestroy cancer cells. The immune effector may be, for example, anantibody specific for some marker on the surface of a tumor cell. Theantibody alone may serve as an effector of therapy or it may recruitother cells to actually effect cell killing. The antibody also may beconjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin Achain, cholera toxin, pertussis toxin, etc.) and serve merely as atargeting agent. Alternatively, the effector may be a lymphocytecarrying a surface molecule that interacts, either directly orindirectly, with a tumor cell target. Various effector cells includecytotoxic T cells and NK cells. The combination of therapeuticmodalities, i.e., inhibition or reduction of PRL-1 expression oractivity would provide therapeutic benefit in the treatment of cancer,such as pancreatic cancer.

Immunotherapy could also be used as part of a combined therapy. Thegeneral approach for combined therapy is discussed herein. In one aspectof immunotherapy, the tumor cell must bear some marker that is amenableto targeting, i.e., is not present on the majority of other cells. Manytumor markers exist and any of these may be suitable for targeting inthe context of the present invention. Common tumor markers which havebeen found to be upregulated in pancreatic cancer include, but are notlimited to carcinoembryonic antigen, CA 27-29 antigen, neuron-specificenolase (NSE), CA 125 antigen, and human chorionic gonadotropin (HCG).

Other types of immunotherapy that may be employed with a PRL-1inhibitor(s) of the present invention are passive and activeimmunotherapy.

A number of different approaches for passive immunotherapy of cancerexist. They may be broadly categorized into the following: injection ofantibodies alone; injection of antibodies coupled to toxins orchemotherapeutic agents; injection of antibodies coupled to radioactiveisotopes; injection of anti-idiotype antibodies; and finally, purging oftumor cells in bone marrow. It may be favorable to administer more thanone monoclonal antibody directed against two different antigens or evenantibodies with multiple antigen specificity. Treatment protocols alsomay include administration of lymphokines or other immune enhancers asdescribed by Bajorin et al. (1988). The development of human monoclonalantibodies is well known to those of skill in the art (see Harlow andLane, 1988)

In active immunotherapy, an antigenic peptide, polypeptide or protein,or an autologous or allogenic tumor cell composition or “vaccine” isadministered, generally with a distinct bacterial adjuvant (Ravindranathand Morton, 1991; Mitchell et al., 1990; Mitchell et al., 1993).

d. Gene Therapy

The present invention also contemplates gene therapy in conjunction withPRL-1 inhibitor therapy. As with the majority of human cancers, numerousgenetic alterations have been identified that play a role inadenocarcinoma of the pancreas. These include mutations in the tumorsuppressor genes p53, Rb, p16, BRCA2 and DPC4. Several activatedoncogenes have also been identified as contributing to pancreas cancerincluding K-ras, HER-2/neu, NFkappaB and AKT2. There are, no doubt, manyother genetic defects that contribute to the onset and progression ofpancreatic cancer and identifying these mutants and the specificconsequences of the defects will lead to a better understanding of howto treat this disease. Gene therapy make also be combined with chemo-and radiotherapy to further improve the efficacy of the inhibitor of thepresent invention. For example, the herpes simplex-thymidine kinase(HS-tK) gene, when delivered to brain tumors by a retroviral vectorsystem, successfully induced susceptibility to the antiviral agentganciclovir (Culver et al., 1992).

Inhibitors of cell proliferation, such as tumor suppressor genes, may beemployed with the PRL-1 inhibitor(s) of the present invention. The tumorsuppressor oncogenes function to inhibit excessive cellularproliferation. The inactivation of these genes destroys their inhibitoryactivity, resulting in unregulated proliferation. The tumor suppressorsp53, p16, Rb, and MMACl/PTEN may be employed with a PRL-1 inhibitor(s)of the present invention in treating a cancer, such as pancreaticcancer. Other genes that may be employed with a PRL-1 inhibitor of thepresent invention include APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II,zac1, p73, VHL, DBCCR-1, FCC, rsk-3, p27, p27/p16 fusions, p21/p27fusions, anti-thrombotic genes (e.g., COX-1, TFPI), PGS, Dp, E2F, ras,myc, neu, raf, erb, fms, trk, ret, gsp, hst, abl, E1A, p300, genesinvolved in angiogenesis (e.g., VEGF, FGF, thrombospondin, BAI-1, GDAIF,or their receptors) and MCC. These genes are provided herein as examplesand are not meant to be limiting.

Genes that regulators of apoptosis, or programmed cell death, may alsobe employed with PRL-1 inhibitor(s) of the present invention in treatingpancreatic cancer. Apoptosis, or programmed cell death, is an essentialprocess for normal embryonic development, maintaining homeostasis inadult tissues, and suppressing carcinogenesis (Kerr et al., 1972). TheBcl-2 family of proteins have been demonstrated, in the art, to beimportant regulators and effectors of apoptosis in numerous systems.Some members of this family e.g., Bax, Bak, Bik, Bim, Bid, Bad,Harakiri, are known to promote cell death and thus may be employed withthe PRL-1 inhibitor(s) of the present invention.

e. Hormonal Therapy

Hormonal therapy may also be used in conjunction with a PRL-1inhibitor(s) of the present invention or in combination with any othercancer therapy described herein. The use of hormones may be employed tolower the level or block the effects of certain hormones that may play arole in the tumor cell proliferation. This treatment is often used incombination with at least one other cancer therapy as a treatment optionor to reduce the risk of metastases in cancers which include but are notlimited to breast, prostate, ovarian, or cervical cancer.

f. Surgery

The present invention may also be used in conjunction with surgery.Surgery may also be used in combination with any of the other cancertherapies described herein such as radiation therapy and chemotherapy.

Surgery may be used to remove all or part of the pancreas. The extent ofsurgery depends on the location and size of the tumor, the stage of thedisease, and the patient's general health. Surgery may employ variousprocedures. One type of surgical procedure that may be use to treatpancreatic cancer is the Whipple procedure. In this procedure, if thetumor is in the head (the widest part) of the pancreas, the surgeonremoves the head of the pancreas and part of the small intestine, bileduct, and stomach. The surgeon may also remove other nearby tissues.Another surgical procedure is a distal pancreatectomy in which thesurgeon removes the body and tail of the pancreas if the tumor is ineither of these parts. A total pancreatectomy may also be performed inwhich the surgeon removes the entire pancreas, part of the smallintestine, a portion of the stomach, the common bile duct, thegallbladder, the spleen, and nearby lymph nodes.

G. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Material and Methods

Microarray Sample Preparation and Hybridization. cDNA microarray slidesused in this study were fabricated in the microarray core facilities atthe Arizona Cancer Center (Calaluce et al., 2001). Briefly, each slidehas 5760 spots divided into four blocks, with each containing eightidentical ice plant genes from Mesembryanthemum crystallinum and 23different housekeeping genes as references for data normalization. Eachslide had 5289 unique human cDNA sequences. Poly(A)⁺ RNA was directlyisolated from cell pellets using the FastTrack 2.0 kit (Invitrogen,Carlsbad, Calif.), following the instruction manual provided by themanufacturer. Normal pancreas Poly(A)⁺ RNA was isolated from total RNA,which was purchased from Clontech Laboratories (Palo Alto, Calif.) usingthe Oligotex Direct mRNA kit (Qiagen, Inc., Valencia, Calif.). This“normal pancreata” consisted of a pool of two tissue specimens donatedby two male Caucasians 18 and 40 years of age. Labeling and purificationof cDNA probes were carried out using the MICROMAX direct cDNAmicroarray system (NEN Life Science Products, Boston, Mass.). Two to 4μg of the Poly(A)⁺ RNA samples were used for each labeling. Probes foreach pancreatic cell line were labeled with cyanine 5 (Cy5), and probesfor HeLa cells were labeled with cyanine 3 (Cy3). For HeLa cell versusnormal pancreas hybridization, a normal pancreas sample was labeled withCy3, and a HeLa cell sample was labeled with Cy5.

Purified cDNA probes were dried and dissolved in 15 μl of hybridizationbuffer (included in the MICROMAX direct cDNA microarray system kit). Theprobes were then denatured by heating at 95° C. for 2 min and applied tothe array area of a predenatured microarray slide. The microarray slidewas covered with a 22×22-cm slide coverslip and incubated in aHybChamber (GeneMachines, San Carlos, Calif.) at 62° C. for overnight.On the second day, the slide was washed in 0.5×SSC, 0.01% SDS for 5 min;0.06×SSC, 0.01% SDS for 5 min; and 0.06×SSC for 2 min. Finally, theslide was dried by spinning at 500×g for 1 min and scanned in adual-laser (635 nm for red fluorescent Cy5 and 532 nm for greenfluorescent Cy3) microarray scanner (GenePix 4000; Axon Instruments,Foster City, Calif.).

RT-PCR. Two μg of total RNA isolated from pancreatic cancer cell pelletsor frozen pancreatic tumor tissues were used for reverse transcriptasereactions (20 μl in total volume), which were carried out using theOmniscript RT kit (Qiagen, Inc.), following the manufacturer's protocol.The PCRs were then carried out by mixing 2 μl of reverse transcriptasereaction mixture, 5 μl of 10×PCR™ buffer containing 15 mM Mg²⁺, 1 μl of10 mM deoxynucleotide triphosphate mixture, 2.5 μl of 5 μM PCR™ primerpair for specific gene, 1 μl of β-actin primer pair, 1 μl of β-actincompetimers (Ambion, Inc., Austin, Tex.), 37 μl of H₂O, and 0.5 μl of 5units/μl Taq polymerase (Promega Corp., Madison, Wis.). Theamplification cycle (94° C. for 30 s; 56° C. for 45 s; and 72° C. for 1min) was repeated 29 times. PCR™ primers for individual genes weredesigned to generate a DNA fragment ˜600 bp in length (if the mRNAitself is less than 600 bases, PCR™ products were generated in maximallength) using the Primer3 program (Rozen and Skaletsky, 2000).

Northern Blot. RNA electrophoresis and transferring to Zeta-Probe GTmembranes (Bio-Rad, Hercules, Calif.) were performed as describedpreviously (Calaluce et al., 2001). ³²P-labeled probes were made fromthe agarose gel-purified RT-PCR products of each gene using the RadPrimeDNA Labeling System (Invitrogen). The probe hybridization and strippingbuffers and conditions were as provided by the membrane manufacturer.Hybridized membranes were exposed to a Phosphorlmager (MolecularDynamics, Sunnyvale, Calif.), and signals were quantified using theImageQuant software.

Pancreatic Tumor Tissue Array Construction and Immunohistochemistry.Morphologically representative areas of 42 archival cases of pancreatictumors, 35 of which are documented ductal adenocarcinomas, from theUniversity of Arizona Health Sciences Center and the Tucson VeteransAdministration Medical Center, are selected from formalin-fixed tissuesamples embedded in paraffin blocks. Two 1.5-mm-diameter cores/case arereembedded in a tissue microarray using a tissue arrayer (BeecherInstruments, Silver Spring, Md.) according to a method describedpreviously (Kononen et al., 1998). Ser. sections of theparaffin-embedded pancreatic tissue array are deparaffinized and reactedwith primary antibodies specific for c-Myc (clone 9E10.3; Neo-Markers,Fremont, Calif.) or Rad51(Oncogene, Boston, Mass.). Before antibodyincubation, the slides are processed for antigen retrieval. This consistof microwaving the slides in citrate buffer (0.1 M, pH 6.0) in apressure cooker for 25 min and then leaving them to cool. The slideswill be incubated with the antibody for 1 h. Biotinylatedanti-mouse/anti-rabbit secondary antibodies are applied, followed bystreptavidin-peroxidase complex (DAKO, Carpinteria, Calif.). Coloredproducts are produced using the diaminobenzidine substrate. Stainingreactions are scored as diffuse or focal and graded (from 0, negative to4+, intensely positive) for both neoplasm and background stroma.

Antisense Experiments. To perform antisense experiments cells (5×10⁵)are incubated in triplicate in 6-well plates in 1 ml of culture mediumsupplemented with 10% heat-inactivated FCS for 30 min at 65° C. todestroy nuclease activity. These cells are then cultured (24 h) in thepresence or absence of antisense, sense, or randomly scrambledphosphorothioate oligonucleotides (ODNs). The ODN sequences are testedagainst sequences in the GenBank™ database. Two distinct antisense ODNscomplementary to sequences that encompass the translation initiationsite of the specific target are used. The ability of the antisense toinhibit expression of the target is verified by RT-PCR and with anantibody for Western blotting. Once it is confirmed that expression ofthe target is downregulated, the phenotypic consequences of inhibitionof the target can be determined using the assays describe below.

Cell Proliferation Assays. Cell proliferation assays with these celllines are performed to determine the effect of target inhibition on cellgrowth. Cells are seeded at 2.0-5.0×10⁵ cells in 100-mm culture dishesand allow to attach overnight at 37° C. Adherent cells are washed andincubated with serum-free RPMI 1640 or RPMI containing 10% FBS for 48 h,after which they are trypsinized and counted using a hemocytometer. Inaddition, parallel experiments are perform and instead of cell counts,the proliferative status of the cell lines is determined using flowcytometric analysis of DNA content. For flow analysis, cells are stainedwith propidium iodide using a modified Krishan technique (Krishan,1975). All samples are analyze with an FACSCAN flow cytometer (BectonDickinson) using a 15 mWatt argon ion laser operated at 6 mWatts ofpower at 488 nm. Photomultiplier tube voltage is adjusted for eachcontrol sample to position the G0/G1 to channel 240 on a 1024 channelpresentation. Histograms are analyze for cell cycle compartments usingCELLQUEST (Becton Dickinson) analysis software. Histograms having 50Kevents are collected to maximize the statistical validity of thecompartmental analysis. The results of this flow analysis allow theexamination of the cell cycle distribution of the pancreatic cancer celllines.

Alternative analysis of proliferative rate can be estimated by a numberof other techniques, including BrdU incorporation or PCNA or Ki67immunostaining, however, flow analysis is preferred, since it providesan estimate of the fraction of cells in the G1 and G2/M stages of thecell cycle as well as in S-phase. By measuring the proliferative statusof the cell lines a better understanding of whether or not the targetplays a role in regulating the growth of pancreatic cancer cells isachieved.

Apoptosis Assays. For the measurement of spontaneous and serumstarvation induced apoptosis before and after target inhibition, thecells are seeded at 2.0-5.0×10⁵ cells in 100-mm culture dishes and allowto attach overnight at 37° C. Adherent cells are washed and incubatedwith serum-free RPMI 1640 media or RPMI 1640 media containing 10% FBSfor 48 hr, at which time they are harvested by trypsinization. Anyfloating cells in the media will be saved and pooled with the harvestedcells for the apoptosis analysis. An annexin V based assay is used toquantitate apoptosis. After initiating apoptosis, cells translocatephosphatidylserine (PS) from the inner face of the plasma membrane tothe cell surface. Once on the cell surface, PS can be detected using aGFP or FITC conjugate of annexin V (Clontech), a protein that has astrong, natural affinity for PS. This simple assay is a one-stepstaining procedure of live cells that takes 10 minutes. After incubationwith the conjugated annexin V the cells are analyzed by flow cytometryand the percentage of labeled cells determined.

Anchorage Dependent Cell Growth. To assess anchorage-independent growth,the target inhibited cells are suspended in reduced-serum (2%) mediumcontaining 0.3% agar, and overlaid onto a 0.6% agar base at a density of2×10⁴ cells/60-mm dish. Colony formation is monitored for up to 1 month.The number of colonies formed by the target inhibited and uninhibitedcells is counted and compared for statistical differences. The abilityof target inhibition to alter anchorage-independency of the pancreaticcancer cell lines is a good indication of whether the target is involvedin promoting tumorigenicity in pancreatic cancer cell lines.

Cell Migration. In addition to anchorage-independent cell growth, therole of target inhibition in suppressing cell migration can be assessed.Multiple signaling pathways are believed to play a role in directed cellmigration. Cell migration is assessed by quantitating the number ofcells that directionally migrate through membranes to a collagenundercoating. Briefly, 1× target inhibited and uninhibited cells areloaded into modified Boyden chambers (tissue culture—treated, 6.5-mmdiameter, 10 μm thickness, 8-μm pores, Transwell®; Costar Corp)containing collagen type I-undercoated membranes. Cells are allowed tomigrate through membranes by incubating them at 37° C. for various timepoints. Nonmigratory cells on the upper membrane surface are removedwith a cotton swab, and the migratory cells attached to the bottomsurface of the membrane are stained with 0.1% crystal violet in 0.1 Mborate, pH 9.0, and 2% ethanol for 20 min at room temperature. Thenumber of migratory cells per membrane is either counted with aninverted microscope using a 40× objective, or the stain is eluted with10% acetic acid and the absorbance at 600 nm determined and migration isenumerated from a standard curve. Differences in the migration capacityof cells between target inhibited and uninhibited cells is evaluated bycomparing the percentages. A decrease in the migration capacityindicates that the target plays a role in regulating cell invasiveness.

Analysis of Gene Expression Patterns. From frozen pancreatic cancerspecimens, frozen tissue sections can be made and examined independentlyof the original pathological report. Total RNA is extracted, using thestandard Triazol RNA isolation protocol (Life Technologies,Gaithersburg, Md.), from tissue blocks that contained over 75% ofneoplastic cells. The amount and the quality of RNA is checked byelectrophoresis on a 1% formamide agarose gel. Normal tissue RNA samplescan be obtained from Clontech (Palo Alto, Calif.). The RNA is labeled byreverse transcription and array hybridizations to the new 10,000-genechip is performed as described above. After analysis, gene expressionpatterns from the frozen tissue are compared to those from the celllines to look for significant differences and for potential new targets.

Example 2 Microarray Analysis

The gene expression patterns of genes from pancreatic cancer cell lineswere analyzed and compared to gene expression in normal pancreas cells.The strategy employed is shown in FIG. 1. Instead of performing straightcomparisons of gene expression in the pancreatic cancer cell lines tonormal pancreas, a universal reference RNA (Hela cell RNA) was used tohybridize to both the cancer cell lines and normal pancreas. The geneexpression ratios were then calculated by dividing out the ratio datafrom the reference as shown in FIG. 1. The reference was used in thisanalysis because it allows for a comparison of multiple hybridizationswhen the control RNA (normal pancreas) is limiting.

Example 3 Hybridization of Expression Products from BXPC-3 PancreaticCancer Cells

FIG. 2 shows a representative array hybridization from a pancreaticcancer cell line hybridized to a reference RNA. To date, gene expressionpatterns from several different pancreatic cancer cell lines have beenanalyzed, and compared to the gene expression patterns of normalpancreas. The probes for the cDNA microarray analysis were made using afluorescent first strand cDNA from 4 μg Poly A+RNA from each of thepancreatic cancer cells in the presence of Cy5-dCTP (Red), and from 4 μgof Poly A+RNA from Hela cells in the presence of Cy3-dCTP (Green). Thetwo fluorescent first strand cDNAs were then mixed, denatured, and usedas targets for the genes on the cDNA microarray slide. Following thehybridization and wash steps, quantitative fluorescent emissions werecollected using a Gene Pix 4000A-microarray reader (Axon Instrunents)and quantitated using the Gene Pix 4000A associated software. The normalpancreas RNA (purchased from Clontech and consisted of pooled RNA from 4different donors) was analyzed in the same way with the Cy5 channelbeing Hela cell RNA and Cy3 channel being normal pancreas. Each cellline was then compared to normal pancreas by simply multiplying theratio data from the cell lines to the normal pancreas. The geneexpression ratio data for each gene was then analyzed and genes showingsignificant changes in gene expression were identified using a 95%confidence interval analysis. Hierarchical cluster analysis was thenused to cluster genes with similar expression patterns into groups. Fromthis analysis, 438 genes were identified as being significantlydownregulated across the pancreas cancer cell lines and 68 genes weresignificantly upregulated. The 68 upregulated genes were screenedfurther for suitability as drug targets. A list of 50 of theseoverexpressed genes, including both known and unknown genes, is shown inTable 1. Examples of potential targets identified by the cDNA microarrayinclude protein tyrosine phosphatase 1 (PRL-1), urokinasetype-plasminogen activator (uPA) and its receptor (uPAR), aurora kinase,CDC28 protein kinase 2, CDC25B and 5′-nucleotidase. TABLE 1 50Overexpressed Genes In Pancreatic Cancer Tissues EphA2 Urokinaseplasminogen activator Non-specific cross reacting PRL-1 phosphataseantigen Thymosin beta PCNA Annexin I (lipocortin I) Human Gu proteinMMP-9 High mobility group (nonhistone) Heparin cofactor II CksHs-2Glutathione peroxidase 2 Dysferlin EVI2A Trinucleotide repeat containing3 GAGH3 ESTs HYPOTHETICAL PROTEIN KIAA0195 Pho GTPase-specific Smallnuclear GTP exchange factor ribonucleoprotein IL1A c-Myc S-100P PROTEINCytochrome c-1 NGAL NUCLEOLYSIN TIA-1 Calgizzarin Human small prolinerich Thioredoxin reductase protein Aurora Kinsase 2 Rho GDP dissociationinhibitor (GDI) beta p33 ING1 DNA primase polypeptide 1 (49 kD) AnnexinVIII CCAAT/enhancer binding protein (C/EBP), beta TROPONIN T RAD51Single-stranded DNA- Met receptor binding protein Keratin 19 NGFInterferon consensus Ciao-1 sequence binding protein 1 Leman coiled-coilprotein EST R23055 FOS-like antigen-1 EST R06944 TRANSCRIPTION ESTR53421 ELONGATION FACTOR SH Plasminogen activator EST R35245 receptor,urokinase receptor

Example 4 Overexpression of PRL-1 in Pancreatic Cell Lines and Tumors

To further analyze and validate the expression patterns of theseoverexpressed genes in pancreatic cancer cell lines, RT-PCR and Northernblotting were used to look at each gene individually. FIG. 3A showsrepresentative data from RT-PCR analysis of some of the genes that areoverexpressed in pancreatic cancer cells versus normal pancreas.

PRL-1 was found to be one of genes that showed the most consistent andsignificant overexpression (Table 2; FIG. 3B). PRL-1 is overexpressed in6 cell lines with a ratio ranging from 3.3 to 9.5. The other 3 celllines did not have an expression ratio recorded because their microarrayhybridization did not pass the quality control. FIG. 3C showsoverexpression of PRL-1 in several patient tumor samples as compared tonormal pancreas. The results from the RT-PCR and Northern blotting haveconfirmed overexpression of the genes from the microarray analysis andboth sets of data correlate well with respect to differences in levelsof expression across the different cell lines. TABLE 2 Expression ratioCell Line (Cell Line/Normal) AsPC-1 4.9 BxPC-3 5.6 Capan-1 N/A CFPAC N/ASu86.86 N/A HPAF II 9.5 Mia Paca-2 4.5 Mutj 3.3 Panc-1 3.7

Example 5 Pancreatic Cancer Tissue Array

In addition to confirming overexpression of the target genes in thepancreatic cancer cell lines, a tissue array was developed that allowsthe determination of the expression of specific gene products in tumorstaken from pancreatic cancer patients (see FIG. 4). The sampling of theoriginal pancreatic cancer tissues for arraying was performed frommorphologically representative regions of formalin-fixedparaffin-embedded tumor and normal tissue blocks. Core tissue biopsies(diameter 0.6 mm, height 3-4 mm) were taken from individual “donor”blocks and arrayed into a new “recipient” paraffin block (45×20 mm)using a tissue microarraying instrument (Beecher Instruments). Onaverage, 200 sections can be cut from one tumor tissue microarray block.HE-staining for histology verification is performed on every 50thsection cut from the block (FIG. 4). Once constructed, the tissuemicroarray slide was then stained using immunohistochemistry withantibodies directed against the proteins of interest and evaluatedeither manually or utilizing a high-throughput digital imaging system.This tissue array system greatly enhances the ability to quicklyvalidate the expression of potential target genes and analyze thefrequency of expression across a number of patient tumors.

Example 6 Effect of Antisense PRL-1 on Inhibiting Pancreatic Cancer CellGrowth

To investigate the effect of PRL-1 inhibition on pancreatic cancer cellgrowth, antisense oligonucleotide studies were conducted. Four antisenseoligonucleotides were designed to target different areas of the PRL-1mRNA. One of these oligonucleofides, AS-Prl-1C, reduced the mRNA levelmore than 90% within 24 hours of treatment (FIG. 5A) and, therefore, waschosen to be used in subsequent studies. A time course treatment of MiaPaCa-2 cells with 200 nM of AS-Prl-1C or the corresponding scramble wasconducted and changes in PRL-1 mRNA level, cell cycle distribution andapoptosis population were examined. The PRL-1 mRNA level was reduced toits lowest level (˜5% of the control) 24 hours after the AS-Prl-1Ctreatment (FIG. 5B). The treatment of Mia PaCa-2 cells with PRL-1antisense oligonucleotides resulted in arrest of cell growth in theG0/G1 phase of the cell cycle. Twenty-four hours after treatment, 85% ofthe cells were in G0/G1 phase and 3% of the cells were in S phasecompared to 60% in G0/G1 and 19% in S phase for the scrambleoligonucleotide treated samples (FIG. 5C). PRL-1 antisenseoligonucleotide treatment also induced apoptosis in the Mia PaCa-2cells. Twelve hours after AS-Prl-1C treatment, the number of apoptoticcells increased dramatically to 40% compared to less than 5% in scrambleoligonucleotide control (FIG. 5D). These results indicated that PRL-1plays a role in cell cycle regulation and that inhibition of PRL-1induces apoptosis in tumor cells.

Example 7 Effect of siRNA Complexes on Inhibiting Pancreatic Cancer CellGrowth

Short interfering RNA (siRNA) has also been used to suppress PRL-1expression in pancreatic cancer cells. In the present invention, doublestranded siRNA complexes are designed using the following guidelines:(1) a double stranded RNA complex is composed of a 21-nucleotide senseand 21-nucleotide anti-sense strand, both with a 2-nucleotide 3′overhang, i.e., a 19 nucleotide complementary region; (2) a 21nucleotide sequence is chosen in the coding region of the mRNA with aG:C ratio as close to 50% as possible, preferably within about 60% toabout 40%, or alternatively within about 70% to about 30%; (3)preferably regions within about 75 nucleotides of the AUG start codon orwithin about 75 nucleotides of the termination codon are avoided; (4)preferably more than three guanosines in a row are avoided as poly Gsequences can hyperstack and agglomerate; (5) preferably choose asequence that starts with AA as this results in siRNA's with dTdToverhangs that are potentially more resistant to nucleases; (6)preferably the sequence is not homologous to other genes to preventsilencing of unwanted genes with a similar sequence. A negative controlmay be included, such a negative control suitably being the samenucleotide sequence as the test siRNA but scrambled such that it lackshomology to any other gene.

Examples of such 21 nucleotide target DNA sequences, and the 19nucleotide sense and antisense sequences utilizing dTdT 3′ overhangs (dTis 2′-deoxythymidine), derived from the coding sequence of PRL-1(derived from GenBank NM_(—)003463[gi:17986281]), include, but are notlimited to, those described in Table 3: TABLE 3 Target RNA Sense RNAAntisense RNA aaacaaauuuauagaggaacu Acaaauuuauagaggaacuttaguuccucuauaaauuugutt (SEQ ID NO:1) (SEQ ID NO:2) (SEQ ID NO:3)aacaaauuuauagaggaacuu Caauuuauagaggaacuutt aaguuccucuauaaauuugtt (SEQ IDNO:4) (SEQ ID NO:5) (SEQ ID NO:6) aaagaagguauccauguucuuAgaagguauccauguucuutt aagaacauggauaccuucutt (SEQ ID NO:7) (SEQ ID NO:8)(SEQ ID NO:9) aaauacgaagaugcaguacaa Auacgaagaugcaguacaattuuguacugcaucuucguautt (SEQ ID NO:10) (SEQ ID NO:11) (SEQ ID NO:12)aagaugcaguacaauucauaa Gaugcaguacaauucauaatt uuaugaauuguacugcauctt (SEQID NO:13) (SEQ ID NO:14) (SEQ ID NO:15) aauucauaagacaaaagcggcUucauaagacaaaagcggctt gccgcuuuugucuuaugaatt (SEQ ID NO:16) (SEQ IDNO:17) (SEQ ID NO:18)

The orientation of the double stranded RNA complex for the firstexemplified sense and antisense siRNA strands in Table 3 is as follows:5′-acaaauuuauagaggaacutt-3′ (SEQ ID NO:2) 3′-ttuguuuaaauaucuccuuga-5′(SEQ ID NO:19)

The above guidelines are solely an aid to designing suitable RNAoligonucleotides and are not a limitation of the interfering RNAoligonucleotides and related methods of use of the present invention.

Thus far, two siRNA sequences have been identified that are able toreduce PRL-1 expression to about 10% of non-treated control (FIG. 6).The phenotypes of the PRL-1 siRNA treated cells are currently underexamination, but it is predicted that the effects of siRNA will besimilar to that seen with PRL-1 antisense oligonucleotides.

Example 8 Screening of Compound Libraries for PRL-1 Inhibitors Using AnEnzymatic Assay

Two different but complementary approaches have been applied to identifynovel small molecular weight inhibitors of PRL-1. One is the highthroughput screen of small molecule libraries using an in vitroenzymatic assay of PRL-1. The other is the PRL-1 homolog model, based onvirtual screening of chemical structure libraries and optimization oflead compounds.

To produce PRL-1 protein for in vitro phosphatase assays, the PRL-1 genewas cloned into an expression vector with a His tag (pcDNA™ 3.1 fromInvitrogen) and the protein expressed in vitro using the TNT coupledtranscription and translation kit (Promega). FIG. 7A shows the Westernblot detection of the His-tagged PRL-1 protein in the TNT mixture. Themolecular weight of this PRL-1 protein (22KDa) is exactly the same asreported in the literature. An in vitro phosphatase assay was conductedusing a tyrosine phosphatase assay system (Promega) to confirm thedephosphorylation activity of the recombinant PRL-1 protein. As shown inFIG. 7B, 20 μl of the dialyzed TNT product increased the phosphataseactivity by 3 times compared to the control (from 0.2 to 0.8 inabsorbance units). Two known tyrosine phosphatase inhibitors, sodiumorthovanadate and EDTA, showed some inhibitory activity against PRL-1(FIG. 7C).

This assay was optimized and used to screen various compound librariesfor PRL-1 inhibitors. FIG. 8 shows the anti-PRL-1 activity of somepositive hits identified from the NCI diversity library and/or theUniversity of Arizona (UA) Natural Products Library. Compoundsidentified using the DiFMUP in vitro assay to screen compounds from theNanosyn Combichem library and the NCI database include NS19999, NS45609,NS45336, and NCI668394 respectively.

Example 9 Molecular Modeling and Virtual Screening of Chemical StructureLibraries

The three-dimensional structure of PRL-1 has not been solved yet.However, structures of various other phosphatases have been published.Given that PRL-1 has about 70% in catalytic domain and 21% overallsequence identity to PTEN, a lipid phosphatase, (FIG. 9) a homologymodel was built based on the PTEN crystal structure (FIG. 10).

Based on this model compound structures from different sources have beendocked to the active site. These structures include the NCI chemicaldatabase, known drug leads for the protein tyrosine phosphatases (PTPs)and inhibitors of the Cdc25B, a dual specfic phosphatase. A molecularspreadsheet was built within the Sybyl. Initially three separatedatabases were generated for input to the virtual docking. From thecombined database a total of 49 structurally diverse compounds wereobtained for further screening. The docking models of three compoundsthat have the best docking to the active site of PRL-1 are shown in FIG.11 (the structures of these compounds are provided in table 5).

The NCI29209 is a substituted 6-methoxy-quinoline class of compoundidentified as PRL-1 inhibitor from high-throughput screening andmolecular modeling methods. The NC129209 compound was obtained from anNCI database and tested on the NCI panel of cell lines for variouscancers. This compound is being utilized as a lead compound foroptimization for design of a novel series of compounds as PRL-1inhibitors. Table 4 also shows a novel series of PRL-1 inhibitorsdesigned using the structure based approach. These compounds are beenanalyzed using enzymatic and cellular assays.

The Nanosyn Combichem library compounds NS12866:[3-(Benzo[1,2,5]-4-sulfonyl-thiadiazole, NS12882:2-Amino-4-trifluoromethanesulfonyl-benzoic acid, as shown in table 5,were modeled using a homology model of PRL-1. Based on FlexX docking thebinding mode of these compounds have been explored and a novel series ofcompounds using a structure-based design strategy are been designed(Table 4). Table 5 also provides a list of PRL-1 inhibitors identifiedby screening various library as well as the IC₅₀ values which for somecompounds was found to be >100 μM. As discussed, several compounds wereidentified by screening the NCI database and the Nanosyn Combichemlibrary however, no hits were obtained from the screening of theLeadQuest (Tripos Inc.) and the MayBridge libraries. TABLE 4 Novel PRl-1Inhibitors

TABLE 5 DOCKING STRUCTURE NAME VALUE IC₅₀

UA78871 −11.4 N/A

UA11656 −4.1 —

UA53892 −7.9 N/A

UA12812 −14.6 66 μM

UA48872 −1.6 N/A

UA97885 0 N/A

UA12499 −14.7 36.5 μM  

UA12690 −8.5 N/A

UA13066 −4.5 —

UA13464 −15.6 72 μM

PTP1B Nova Nordisk NNC-52-1236 −26.2 N/A

Abbott-10 A-366901 −19.8 N/A

Abbott A-321842 −14.0 N/A

Korea Research Institute of Chem. Tech. 1,2-naphtoquinone derivatives−8.9 N/A

Albert Einstein College of Med. 4′-phosphonyldifluoromethyl-phenylanaline derivatives −13.9 N/A

Merck-Frosst methylphosphonc acid derivatives −14.0 N/A

Aventis Benzooxathiazole derivatives −20.8 N/A

Ontogen Cinnamic acid derivatives −20.1 N/A

Japan Tobacco Hydroxyphenyl azole derivatives −8.4 N/A

Takeda Pyrrol phenoxy propionic acid derivatives −8.6 N/A

Molecumetics Phenylatanine derivatives −13.6 N/A

Pharmacia 3′-caxboxy-4′ (O- carboxymethyl)- tyrosine derivatives −27.7N/A

Wyeth Ertiprotafib (Phase II discontinued) −16.0 N/A

Sugen Trifluoramethyl sulfonyl derivatives −24.0 N/A

UA292O9 and/or NCI29209 −14.2 41 μM

UA12882 and/or NS12882 −22.9 25 μM

UA12866 and/or NS12866 −23.6 N/A

UA668394 and/or NC1668394 −11.2 7.2 μM 

UA668394-1

UA668394-2

R₁ = H, F, Cl R₂ = F, Cl, Br

R₁ = H, F, Cl R₂ = F, Cl, Br

UA13378 R/S −14.1 N/A

UA13082 −14.4 8.7 μM 

UA14798 −8.5 64 μM

UA16551 −11.0 N/A

UA339585 −8.8 N/A

UA19999 and/or NS199999 μ17.1 33 μM

UA21497 −20.3 0.1 μM 

UA45336 and/or NS45336 −20.3 12.5 μM 

UA45609 and/or NS45609 −17.8 17 μM

Example 10 Lipid Phosphatase Activity of PRL-1

Since the molecular modeling study showed that PRL-1 shares a similarstructure with the lipid phosphatase PTEN, PRL-1 was tested for possiblelipid phosphatase activity. The results are rather intriguing. As shownin FIG. 12, PRL-1 exhibited very strong lipid phosphatase activitycompared to its PTPase activity. Most interestingly, the lipidphosphatase activity of PRL-1 is specific to 4-phosphate. PRL-1 producedfree phosphate when phosphatidylinositol 3,4,5-trisphosphate (PI3,4,5-P₃), PI 3,4-P₂, and PI 4,5-P₂ were used as substrates but failedto do so when PI 3,5-P₂ Was used as substrate (FIG. 12). This activityis different from that of PTEN which is an inositol 3-phosphatase. It iswell known that 3-phosphatases and 5-phosphatases are key players in theinsulin signaling pathway. The significance of this 4-phosphataseactivity of PRL-1 remains to be studied.

Example 11 Analogs for the Inhibition of PRL-1 Phosphatase

UA668394 was previously identified as a PRL-1 phosphatase inhibitor(IC₅₀=7 μM) using high throughout library screening. UA668394 was foundto inhibit the growth of the pancreatic cancer cell line MiaPaCa-2 at anIC₅₀ of 1.2 μM, using a MTS cell proliferation assay (FIG. 13A). Toobtain more potent inhibitors of PRL-1 with higher anti-pancreaticcancer activities, a series of analogues of UA668394 were used (Table5).

Analogs of UA668394 were identified and synthesized Further studies wereconducted to determine the ability of these compounds to inhibit cellproliferation in pancreatic cancer cells. Pancreatic cancer cells,Panc-1 and Mia PaCa-2, were treated with UA668394-1 and UA66839-2analogs, as described above. The results showed that compared toUA668394, the UA668394-1 analog was a better PRL-1 inhibitor compound.Specifically, UA668394-1 (HT-8) has submicromolar IC₅₀ against MiaPaCa-2and Panc-1 pancreatic cancer cells (0.5 μM and 0.7 μM,respectively)(FIG. 13B), while UA668394-2 (HT-11), an isomer ofUA668394, showed very similar activity to that of UA668394 (IC₅₀=2.2 μMin MiaPaCa-2 cells)(FIG. 13C). The fluorine or chloride substitutedcompounds (see Table 5) are designed to lower the molecular weight,increase the bioavailability and lower the non-specific binding. Allcompounds are evaluated in a cell free PRL-1 assay for enzymaticinhibition.

Example 12 Purification of Recombinant PRL-1 Protein Using Ni-NTA Column

To produce active PRL-1 protein for in vitro PRL-1 phosphatase assays,Invitrogen's Ni-NTA Purification System (Invitrogen), was used to purifyrecombinant PRL-1 protein expressed in bacteria.

The inventors first cloned the full length open reading frame of PRL-1to the bacteria expression vector pProEx-HTa (Invitrogen) under thecontrol of an IPTG inducible promoter. A six-histidine tag was added tothe C-terminus of PRL-1 for quick purification of PRL-1 using the Ni-NTAsystem. The expression vector was transformed to bacteria strain BL21and checked for PRL-1 expression using Western blot. For large scaleexpression, bacteria were grown in 500 ml LB media to logarithm phase(OD₆₀₀ between 0.5 to 0.9) and induced to express PRL-1 by adding IPTGto final concentration of 1 mM and incubating for 4 hours. Bacteria wereharvested by centrifugation at 5,000 rpm for 5 min and resuspended inthe Native Binding Buffer (50 mM NaPO4, 0.5M NaCl, pH8.0, and 10 mMimidazole) at 16 ml/100 ml culture. The bacteria cells were then lysatedby adding lmg/ml lysozyme and sonication. The cell lysate wascentrifuged at 3,000 g for 15 min and the supernatant were thentransferred to a 10-ml column pre-packed with 1.5 ml of Ni-NTA resin(Invitrogen). The column was gently agitated for 60 min to allow thebinding of the His-tagged PRL-1 to the resin. After the bindingreaction, the column was washed with the Native Wash Buffer (50 mMNaPO4, 0.5M NaCl, pH 8.0, and 20 mM imidazole) for 4 times. Finally, thePRL-1 protein was eluted off the column with 10 ml of Native ElutionBuffer (50 mM NaPO4, 0.5 M NaCl, pH 8.0, and 250 mM imidazole). 0.5 mlfractions were collected and analysed by SDS-PAGE. The fractionscontaining the PRL-1 protein were combined and stored at 4° C. or −20°C. with the addition of 30% glycerol. The concentration of the proteinwas estimated by measuring the absorbance at OD₂₈₀ using aspectrophotometer. The activity of the protein was evaluated by theenzymatic PRL-1 assay.

Example 13 Inhibition of PRl-1 Expression by siRNA

siRNA oligonucleotides specific to the PRL-1 mRNA were used to suppressthe expression of PRL-1 gene. To achieve maximal suppression, a mixtureof 4 siRNA oligonucleotides that target different regions of the PRL-1mRNA were used. These siRNA were designed and synthesized by theDharmacon RNA Technologies (Lafayette, CO). Each of the siRNAoligonucleotide duplexes was denatured and annealed individually beforebeing mixed together in equal moles to form a siRNA oligonucleotide pool(SMARTPool, Dharmacon RNA Technologies). A stock solution of 20 μM wasprepared and stored at −20° C.

A transient transfection procedure was used to evaluate the inhibitionof PRL-1 expression by the SMARTPool siRNA mixture. Briefly, MiaPaCa-2cells were grown to 40-50% confluency the day of transfection in 6-wellplates and washed with Dulbecco's phosphate bufferd saline (PBS buffer,Cellgro, Herdon, Va.). OPTI-MEM transfection media (Introgen, Carlsbad,Calif.) containing 3 μl of Lipofectin reagent (Invitrogen) per ml ofmedia for each 100 nanomoles of siRNA oligonucleotides used was added tothe cell culture plates. siRNA oligonucleotides were then added dropwiseto obtain the final concentrations. Cells were incubated in transfectionmedia for 6 hours, then washed once with PBS and given normal growthmedia. Cells were harvested with trypsinization. To evaluate the PRL-1expression levels in the siRNA treated cells, total RNA was isolatedfrom the harvested cells using SNAP RNA isolation kit (Invitrogen) andRT-PCR was carried out using the Omniscript RT kit (Qiagen, Valencia,Calif.). The β-actin transcript was also amplified in each reaction toserve as an internal control. As shown in FIG. 14, 72 hours pasttransfection the siRNA oligos suppressed expression of PRL-1 more than90% at all three concentrations tested (50, 100 and 200 nM in lanes 6, 7and 8 of FIG. 14).

Example 14 PTEN Assay

To further identify PRL-1 inhibitors the University of Arizona (UA)Natural Products Library was screened in a similar manner to thatdiscussed above in Examples 8 and 9. Five additional PRL-1 inhibitorswere identified (Table 6). Since the molecular modeling study showedthat PRL-1 shares a similar structure with PTEN, as discussed in Example10, these compounds were tested for PTEN activity. PTEN is an inositol3-phosphatase which cleaves a phosphate from PI(3,4,5)P3. Studies wereconducted to confirm that the compounds identified are specific forPRL-1 activity and not PTEN activity. Thus, a PTEN assay was conductedusing malachite green as the substrate. Malachite green is known to forma complex with free phosphate. The plates were read at 630 nm using aspectrophotometer. As shown in FIG. 15, these compounds did not exhibitPTEN activity when compared to the DMSO control and sodium orthovandateNAVO₄ a positive control.

Next, the compounds identified were tested for their ability to inhibitPRL-1 activity. The UA64859, UA47548, UA63415 compounds exhibited 76%,70% and 62% PRL-1 inhibitory activity, respectively. On the other hand,the least PRL-1 inhibition was observed with the UA61880 (50%inhibition) and UA58428 (42% inhibition) compounds. TABLE 6 STRUCTURENAME INHIBITION %

UA47548 70%

UA58428 42%

UA61880 50%

UA63415 62%

UA64859 76%

Example 15 Cell Proliferation by PRL-1 Inhibitors

Compounds identified as being positive for anti-PRL-1 activity weretested for their ability to inhibit cell proliferation in humanpancreatic cancer cells. The inhibitory activity of UA668394, UA19999and UA45336 (Table 5) were examined. Cell proliferation assays wereconducted using the pancreatic cancer cell line Mia PaCa-2, as describedin Example 1. Briefly, 2.0-5.0×10⁵ cells were seeded in 100-mm culturedishes and allowed to attach overnight at 37° C. Adherent cells werewashed and incubated with serum-free RPMI 1640 or RPMI containing 10%FBS for 48 h, and treated with the compound of interest to determine itseffect on the inhibition of cell growth. For example, cells were treatedwith of the UA668394, UA19999 and UA45336 compounds and analyzed forinhibition of cell proliferative using a MTS assay. The data shows thatthe UA668394 compound was better at inhibiting cell proliferation thanthe UA19999 or UA45336 compounds. Specifically, the IC₅₀ of Mia PaCa-2cells treated with the UA668394 compound was found to be 1.2 μM whereas,cells treated with the UA19999 and UA45336 compounds showed an IC₅₀ of120 μM and 95 μM respectively (FIG. 16A). Thus, in Mia PaCa-2 pancreaticcancer cells the UA668394 compound was found to have the best overallinhibition of cell proliferation.

Example 16 MiaPaca Human Pancreatic In Vivo Xenograft Model

The antitumor effect of PRL-1 is assessed against the MiaPaca humanpancreatic tumor model. MiaPaca tumors are implanted subcutaneously intothe flanks of nude mice. As the tumors reach a predetermined size ofapproximately 100 mm³, the mice are randomized into therapy groups.UA668394-1 is administered by IV injection given for 5 daily doses atmaximum tolerated dose (MTD), ½ MTD, ¼ MTD. Mean tumor volume aredetermined three times per week. Tumor volume is determined by calipermeasurements (mm) and using the formula for an ellipsoid sphere:L×W²/2=mm³, where L is the length in mm and W is the width in mm. Theformula is also used to calculate tumor weight (mg), assuming unitdensity (1 mm³=1 mg). The study is terminated when the tumor volumes inthe control group(s) reach 2000 mm³. The time to reach evaluation sizefor the tumor of each animal is used to calculate the overall delay inthe growth of the median tumor (T-C).

All of the compositions and/or methods and/or apparatus disclosed andclaimed herein can be made and executed without undue experimentation inlight of the present disclosure. While the compositions and methods ofthis invention have been described in terms of preferred embodiments, itwill be apparent to those of skill in the art that variations may beapplied to the compositions and/or methods and/or apparatus and in thesteps or in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   U.S. Pat. No. 4,367,110-   U.S. Pat. No. 4,415,732-   U.S. Pat. No. 4,452,901-   U.S. Pat. No. 4,458,066-   U.S. Pat. No. 5,221,605-   U.S. Pat. No. 5,238,808-   U.S. Pat. No. 5,279,721-   U.S. Pat. No. 5,310,687-   U.S. Pat. No. 5,354,855-   U.S. Pat. No. 5,795,715-   U.S. Pat. No. 5,889,136-   Bajorin et al., J. Clin. Oncol., 6(5):786-92, 1988.-   Berzal-Herranz et al., Genes Dev., 6(1):129-134, 1992.-   Bosher and Labouesse, Nat. Cell. Biol, 2:E31-E36, 2000.-   Calaluce et al., Mol. Carcinog., 30:119-129, 2001.-   Caplen et al., Gene, 252(1-2):95-105, 2000.-   Cates et al., Cancer Lett., 110(1-2):49-55, 1996.-   Cech et al., Cell, 27(3 Pt 2):487-496, 1981.-   Chowrira et al., J. Biol. Chem., 268:19458-62, 1993.-   Chowrira et al., J. Biol. Chem., 269(41):25856-25864, 1994.-   Culver et al., Science, 256(5063):1550-1552, 1992.-   De Jager et al., Semin. Nucl. Med., 23(2):165-179, 1993.-   Diamond et al., Am. J. Physiol., 271(1-1):G121-9, 1996.-   Diamond et al., Mol. Cell. Biol., 14(6):3752-3762, 1994.-   Ditzel et al., Proc. Natl. Acad. Sci. USA, 94:8110-8115, 1997.-   Doolittle and Ben-Zeev, Methods Mol Biol., 109:215-237, 1999.-   Elbashir et al., Genes Dev., 5(2):188-200, 2001.-   Elbashir et al., Nature, 411(6836):494-498, 2001.-   Elchebly et al., Science, 283(5407):1544-1548, 1999.-   Fire et al., Nature, 391:806-811, 1998.-   Forster and Symons, Cell, 49(2):211-220, 1987.-   Grishok et al., Science, 287:2494-2497, 2000.-   Gulbis and Galand, Hum Pathol, 24(12):1271-85, 1993.-   Harlow and Lane, Antibodies: A Laboratory manual, Cold Spring Harbor    Laboratory, 1988.-   Haseloff and Gerlach, Nature, 334:585-591, 1988.-   Hu et al., Oncology, 64:160-165, 2003.-   Joyce, Nature, 338:217-244, 1989.-   Kerr et al., Br. J. Cancer, 26(4):239-257, 1972.-   Ketting et al., Cell, 99:133-141, 1999.-   Kim and Cech, Proc. Natl. Acad. Sci. USA, 84:8788-8792,1987.-   Kononen et al., Nat. Med., 4(7):844-847,1998.-   Krishan, J. Cell Biol., 66(1):188-93, 1975.-   Lieber and Strauss, Mol. Cell. Biol., 15: 540-551, 1995.-   Lin and Avery, Nature, 402:128-129, 1999.-   Luttges et al., Histpathol., 32:444-448, 1998.-   Martin et al, Exp. Hematol, 26:252-264, 1998.-   Michel and Westhof, J. Mol. Biol., 216:585-610,1990.-   Mitchell et al., Ann. NY Acad. Sci., 690:153-166, 1993.-   Mitchell et al., J. Clin. Oncol., 8(5):856-869, 1990.-   Montgomery et al., Proc. Natl. Acad. Sci. USA, 95:155-2-15507, 1998.-   Nakamura et al., In: Handbook of Experimental Immunology (4^(th)    Ed.), Weir et al., (eds). 1:27, Blackwell Scientific Publ., Oxford,    1987.-   Palukaitis et al., Virology, 99:145-151, 1979.-   Patani and LaVoie, Chem. Rev., 96:3147-3178, 1996.-   PawsonTrends Genet., 17(11-12):343-5, 1994.-   PCT Appl. WO 00/44914-   PCT Appl. WO 01/36646-   PCT Appl. WO 01/68836-   PCT Appl. WO 84/03564-   PCT Appl. WO 99/32619-   Perriman et al., Gene, 113:157-163,1992.-   Perrotta and Been, Biochemistry, 31(1):16-21, 1992.-   Prody et al., Science, 231:1577-1580, 1986.-   Rafie et al., Pancreas, 7:123-131, 1992.-   Ravindranath and Morton, Intern. Rev. Immunol., 7: 303-329, 1991.-   Reinhold-Hurek and Shub, Nature, 357:173-176, 1992.-   Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,    1990.-   Rozen and Skaletsky, Methods Mol. Biol., 132:365-386, 2000.-   Sambrook et al., In: Molecular cloning, Cold Spring Harbor    Laboratory Press, Cold Spring Harbor, 2001.-   Sarver et al., Science, 247:1222-1225, 1990.-   Scanlon et al., Proc. Natl. Acad. Sci. USA, 88:10591-10595, 1991.-   Sharp and Zamore, Science, 287:2431-2433, 2000.-   Sharp, Genes Dev., 13:139-141, 1999.-   Sioud et al., J. Mol. Biol., 223:831-835, 1992.-   Symons, Annu. Rev. Biochem., 61:641-671, 1992.-   Tabara et al., Cell, 99:123-132, 1999.-   Thompson et al., Nature Genet., 9:444-450, 1995.-   Tonks et al., Cell, 87(3):365-368, 1996.-   Wang et al., Cancer, 88:2787-2795, 2000.-   Wang et al., J. Biol. Chem., 277(48):46659-46668, 2002.-   Wincott et al., Nucleic Acids Res., 23(14):2677-2684, 1995.-   Yuan and Altman, Science, 263:1269-1273, 1994.-   Yuan et al., Proc. Natl. Acad. Sci. USA, 89:8006-8010, 1992.-   Zeng et al., Biochem. Biophys Res. Commun., 244(2):421-427, 1998.

1. A method of diagnosing or predicting development of pancreatic cancerin a subject comprising: (a) obtaining a cell-containing sample fromsaid subject; and (b) assessing PRL-1 activity or expression in a cellof said cell sample, wherein increased activity or expression of PRL-1in said cell, when compared to a normal cell of the same type, indicatesthat said subject has or is at risk of developing pancreatic cancer. 2.The method of claim 1, wherein said cell is a tumor cell.
 3. The methodof claim 1, wherein assessing comprises assessing PRL-1 expression. 4.The method of claim 3, wherein assessing PRL-1 expression comprisesNorthern blotting.
 5. The method of claim 3, wherein assessing PRL-1expression comprises quantitative RT-PCR.
 6. The method of claim 3,wherein assessing PRL-1 expression comprises Western blotting.
 7. Themethod of claim 3, wherein assessing PRL-1 expression comprisesquantitative immunohistochemistry.
 8. The method of claim 1, whereinassessing comprises assessing PRL-1 activity.
 9. The method of claim 1,wherein said subject has previously been diagnosed with cancer.
 10. Themethod of claim 1, wherein said subject has not previously beendiagnosed with cancer and appears cancer free at the time of testing.11. The method of claim 1, further comprising administering aprophylactic cancer treatment to said subject following testing.
 12. Themethod of claim 1, further comprising administering a cancer therapy tosaid subject following testing.
 13. The method of claim 12, wherein saidcancer therapy is a chemotherapy, a radiotherapy, an immunotherapy, agene therapy, a hormonal therapy or surgery.
 14. A method of predictingthe efficacy of a cancer therapy comprising: (a) administering a cancertherapy to said subject; (b) obtaining a tumor cell-containing samplefrom said subject; and (c) assessing PRL-1 activity or expression in atumor cell of said sample, wherein decreased activity or expression ofPRL-1 in said tumor cell, when compared to a tumor cell of the same typeprior to treatment, indicates that said therapy is efficacious.
 15. Themethod of claim 14, wherein assessing PRL-1 expression comprisesmeasuring PRL-1 protein levels.
 16. The method of claim 14, whereinassessing PRL-1 expression comprises measuring PRL-1 transcript levels.17. The method of claim 14, further comprising assessing PRL-1 activityor expression at multiple time points.
 18. A method of screening acandidate compound for anti-cancer activity comprising: (a) providing acell; (b) contacting said cell with a candidate compound; and (c)assessing the effect of said candidate compound on PRL-1 expression oractivity, wherein a decrease in the amount of PRL-1 expression oractivity, as compared to the amount of PRL-1 expression or activity in asimilar cell not treated with said candidate compound, indicates thatsaid candidate compound has anti-cancer activity.
 19. The method ofclaim 18, wherein said candidate compound is a protein, a nucleic acidor a organo-pharmaceutical.
 20. The method of claim 18, wherein saidcell is a tumor cell.
 21. The method of claim 18, wherein assessingcomprises assessing PRL-1 expression.
 22. The method of claim 21,wherein assessing PRL-1 expression comprises Northern blotting.
 23. Themethod of claim 21, wherein assessing PRL-1 expression comprisesquantitative RT-PCR.
 24. The method of claim 21, wherein assessing PRL-1expression comprises Western blotting.
 25. The method of claim 21,wherein assessing PRL-1 expression comprises quantitativeimmunohistochemistry.
 26. The method of claim 18, wherein assessingcomprises assessing PRL-1 activity.
 27. A method of treating cancercomprising administering to a subject in need thereof a composition thatinhibits PRL-1 activity.
 28. The method of claim 27, wherein saidcompound inhibits PRL-1 expression.
 29. The method of claim 27, whereinsaid candidate compound is a protein, a nucleic acid or anorgano-pharmaceutical.
 30. The method of claim 29, wherein said proteinis an antibody that binds immunologically to PRL-1.
 31. The method ofclaim 29, wherein said nucleic acid is a PRL-1 antisense nucleic acid, aPRL-1 RNAi nucleic acid, or an antibody encoding a single-chain antibodythat binds immunologically to PRL-1.
 32. The method of claim 27, whereinsaid cancer is selected from the group consisting of pancreatic cancer,leukemia, ovarian cancer, breast cancer, lung cancer, colon cancer,liver cancer, prostate cancer, testicular cancer, stomach cancer, braincancer, bladder cancer, head & neck cancer, and melanoma.
 33. The methodof claim 27, further comprising administering a second cancer therapy tosaid subject.
 34. The method of claim 33, wherein said second cancertherapy is a chemotherapy, a radiotherapy, an immunotherapy, a genetherapy, a hormonal therapy or surgery.
 35. The method of claim 27,wherein said composition is administered more than once.
 36. A method ofdiagnosing or predicting development of pancreatic cancer in a subjectcomprising subjecting said subject to whole body scanning for PRL-1activity or expression in a cell.
 37. A method of monitoring ananticancer therapy comprising assessing the expression or function ofPRL-1 in a cancer cell of a subject following or during provision ofsaid anticancer therapy.